Peptide nucleic acids complexes of two peptide nucleic acid strands and one nucleic acid strand

ABSTRACT

Peptide nucleic acids and analogues of peptide nucleic acids are used to form duplex, triplex, and other structures with nucleic acids and to modify nucleic acids. The peptide nucleic acids and analogues thereof also are used to modulate protein activity through, for example, transcription arrest, transcription initiation, and site specific cleavage of nucleic acids.

RELATED APPLICATION

This patent application is a division of patent application Ser. No.08/88,658, filed Jul. 2, 1993 (now U.S. Pat. No. 5,641,625), which is acontinuation-in-part of patent application Ser. No. 08/054,363, filedApr. 26, 1993 (now U.S. Pat. No. 5,539,082), which is acontinuation-in-part of application PCT EP92/01219, filed May 19, 1992(now abandoned) and published Nov. 26, 1992 as WO 92/20702.

FIELD OF THE INVENTION

This invention is directed to compounds that form triple-strandedstructures with single-stranded and double-stranded nucleic acids. It isfurther directed to the use of such compounds to cause stranddisplacement in double-stranded nucleic acids. The invention further isdirected to processes for modifying double-stranded nucleic acidutilizing such strand displacement. Such processes for modifyingdouble-stranded nucleic acids include cleavage of the nucleic acidstrand or strands. In particular, such cleavage includes sequencespecific cleavage of double-stranded nucleic acids using a nucleasewhich normally is nonsequence specific. Such processes also includetranscription inhibition or arrest as well as transcription initiation.The processes of the invention are effected, in particular, withcompounds that include naturally-occurring nucleobases or othernucleobase-binding moieties covalently bound to a polyamide backbone.

BACKGROUND OF THE INVENTION

The function of a gene starts by transcription of its information to amessenger RNA (mRNA) which, by interaction with the ribosomal complex,directs the synthesis of a protein coded for by the mRNA sequence. Thesynthetic process is known as translation. Translation requires thepresence of various co-factors and building blocks, the amino acids, andtheir transfer RNAs (tRNA), all of which are present in normal cells.

Transcription initiation requires specific recognition of a promoter DNAsequence by the RNA-synthesizing enzyme, RNA polymerase. In many casesin prokaryotic cells, and probably in all cases in eukaryotic cells,this recognition is preceded by sequence-specific binding of a proteintranscription factor to the promoter. Other proteins which bind to thepromoter, but whose binding prohibits action of RNA polymerase, areknown as repressors. Thus, gene activation typically is regulatedpositively by transcription factors and negatively by repressors.

Most conventional drugs function by interaction with and modulation ofone or more targeted endogenous proteins, e.g., enzymes. Typical dailydoses of drugs are from 10⁻⁵ -10⁻¹ millimoles per kilogram of bodyweight or 10⁻³ -10 millimoles for a 100 kilogram person. If thismodulation instead could be effected by interaction with andinactivation of mRNA, a dramatic reduction in the necessary amount ofdrug necessary likely could be achieved, along with a correspondingreduction in side effects. Further reductions could be effected if suchinteraction could be rendered site-specific. Given that a functioninggene continually produces mRNA, it would thus be even more advantageousif gene transcription could be arrested in its entirety.

Synthetic reagents that bind sequence selectively to single-stranded andespecially to double-stranded nucleic acids are of great interest inmolecular biology and medicinal/chemistry, since such reagents mayprovide the tools for developing gene-targeted drugs and othersequence-specific gene modulators. Until now oligonucleotides and theirclose analogues have presented the best candidates for such reagents.

Oligodeoxyribonucleotides as long as 100 base pairs (bp) are routinelysynthesized by solid phase methods using commercially available, fullyautomatic synthesis machines. oligoribonucleotides, however, are muchless stable than oligodeoxyribonucleotides, a fact which has contributedto the more prevalent use of oligodeoxyribonucleotides in medical andbiological research directed to, for example, gene therapy and theregulation of transcription and translation. Syntheticoligodeoxynucleotides are being investigated for used as antisenseprobes to block and eventually breakdown mRNA.

It also may be possible to modulate the genome of an animal by, forexample, triple helix formation using oligonucleotides or other DNArecognizing agents. However, there are a number of drawbacks associatedwith oligonucleotide triple helix formation. For example, triple helixformation generally has only been obtained using homopurine sequencesand requires unphysiologically high ionic strength and low pH. Whetherused as antisense reagents or a triplexing structures, unmodifiedoligonucleotides are unpractical because they have short in vivohalf-lives. To circumvent this, oligonucleotide analogues have beenused.

These areas for concern have resulted in an extensive search forimprovements and alternatives. For example, the problems arising inconnection with double-stranded DNA (dsDNA) recognition through triplehelix formation have been diminished by a clever "switch back" chemicallinking whereby a sequence of polypurine on one strand is recognized,and by "switching back", a homopurine sequence on the other strand canbe recognized. See, e.g., McCurdy, Moulds, and Froehler, Nucleosides, inpress. Also, helix formation has been obtained by using artificialbases, thereby improving binding conditions with regard to ionicstrength and pH.

In order to improve half-life as well as membrane penetration, a largenumber of variations in polynucleotide backbones has been undertaken.These variations include the use of methylphosphonates,monothiophosphates, dithiophosphates, phosphoramidates, phosphateesters, bridged phosphoramidates, bridged phosphorothioates, bridgedmethylenephosphonates, dephospho internucleotide analogs with siloxanebridges, carbonate bridges, carboxymethyl ester bridges, acetamidebridges, carbamate bridges, thioether, sulfoxy, sulfono bridges, various"plastic" DNAs, α-anomeric bridges, and borane derivatives. The greatmajority of these backbone modifications have decreased the stability ofhybrids formed between a modified oligonucleotide and its complementarynative oligonucleotide, as assayed by measuring T_(m) values.Consequently, it is generally believed in the art that backbonemodifications destabilize such hybrids, i.e., result in lower T_(m)values, and should be kept to a minimum.

The discovery of sequence specific endonucleases (restriction enzymes)was an essential step in the development of biotechnology, enabling DNAto be cut at precisely specified locations containing specific basesequences. However, although the range of restriction enzymes now knownis extensive, there is still a need to obtain greater flexibility in theability to recognize particular sequences in double-stranded nucleicacids and to cleave the nucleic acid specifically at or about therecognized sequence.

Most restriction enzymes recognize quartet or sextet DNA sequences andonly a very few require octets for recognition. However, restrictionenzymes have been identified and isolated only for a small subset of allpossible sequences within these constraints. A need exists, especiallyin connection with the study of large genomic DNA molecules, in general,and with the human genome project, in particular, to recognize andspecifically cleave DNA molecules at more rarely occurring sites, e.g.,sites defined by about fifteen base pairs.

Efforts have therefore been made to create artificial "restrictionenzymes" or to modify the procedures for using existing restrictionenzymes for this purpose. Methods investigated include the developmentof oligonucleotides capable of binding sequence specifically via triplehelix formation to double-stranded DNA tagged with chemical groups(e.g., photochemical groups) able to cleave DNA or with non-specific DNAcleaving enzymes and other such modifications consistent with an"Achilles heel" general strategy. Such methods are described by:Francois, J. C., et al. PNAS 86,9702-9706 (1989); Perrouault, L., et alNature 344,358-360 (1990); Strobel, S. A. & Dervan P. B. Science249,73-75 (1990); Pei, D., Corey D. R. & Schultz P. G. PNAS 87,9858(1990); Beal, P. A. & Dervan P. B., Science 251,1360 (1991); Hanvey, J.C., Shimizu M. & Wells R. D. NAR 18,157-161 (1990); Koob, H. & SzybalskiW. Science 250,271 (1990); Strobel, S. A. & Dervan P. B. Nature #50,172(1991); and Ferrin, L. J. & Camerini-Otero R. D. Science 254,1494-1497(1991).

In Patent Cooperation Treaty Applications No. PCT/EP92/01220, andPCT/EP92/01219, both filed on May 22, 1992, we described certain nucleicacid analogue compounds that have a strong sequence specific DNA bindingability. Examples of such compounds were also disclosed by us in Science1991, 254 1497-1500. We have shown that a nucleic acid analogue of thistype containing 10 bases hybridized to a non-terminal region of adouble-stranded DNA and rendered the strand of DNA which wasnon-complementary to the nucleic acid analogue susceptible todegradation by S₁ nuclease. No increased cleavage of the DNA strandcomplementary to the nucleic acid analogue was seen, so no cleavage ofdouble-stranded DNA was obtained.

OBJECTS OF THE INVENTION

It is an object of the invention to provide compounds that bind DNA andRNA strands.

It is a further object of the invention to provide triplex structuresbetween DNA or RNA strands and these compounds.

It is yet another object to provide compounds other than RNA that canbind one strand of a double-stranded polynucleotide, thereby displacingthe other strand.

It is still another object to provide therapeutic, diagnostic, andprophylactic methods that employ such compounds.

BRIEF DESCRIPTION OF THE INVENTION

In the cell, DNA exists as a double stranded structure. During certaincellular events, as for instance transcription or during cell division,portions of the double stranded DNA are transiently denatured to singlestrand. Further DNA can be isolated outside of a cell and can bepurposefully denatured to single stranded DNA. RNA generally exist as asingle stranded structure; however, in a local area of secondarystructure a RNA, as for instance the stem of a stem loop structure, theRNA can exist as a double stranded structure.

We have found that certain compounds that have nucleobases attached toan aminoethylglycine backbone and other like backbones includingpolyamides, polythioamides, polysulfinamides and polysulfonamides, whichcompounds we call peptide nucleic acids or PNA, surprisingly bindstrongly and sequence selectively to both RNA and DNA.

We have surprisingly found that these PNA compounds recognize and bindsequence-selectively and strand-selectively to double-stranded DNA(dsDNA). We have found that the binding to double-stranded DNA isaccomplished via strand displacement, in which the PNA binds viaWatson-Crick binding to its complementary strand and extrudes the otherstrand in a virtually single-stranded conformation. We have alsosurprisingly found that these PNA compounds recognize and bindsequence-selectively to single-stranded DNA (ssDNA) and to RNA.

The recognition of PNA to RNA, ssDNA or dsDNA can take place insequences at least 5 bases long. A more preferred recognition sequencelength is 5-60 base pairs long. Sequences between 10 and 20 bases are ofparticular interest since this is the range within which unique DNAsequences of prokaryotes and eukaryotes are found. Sequences of 17-18bases are of special interest since this is the length of uniquesequences in the human genome.

We have further surprisingly found that the PNA compounds are able toform triple helices with dsDNA. We have found that PNA compounds areable to form triple helices with RNA and ssDNA. The resulting triplexes,e.g., (PNA)₂ /DNA or (PNA)₂ /RNA, surprisingly have very high thermalstability. It has been found that the PNA binds with a DNA or RNA ineither orientation, i.e., the antiparallel orientation where theamino-terminal of the PNA faces the 3' end of the nucleic acid or theparallel orientation where the amino-terminal of the PNA faces the 5'end of the nucleic acid. PNAs are able to form triple helices wherein afirst PNA strand binds with RNA or ssDNA and a second PNA strand bindswith the resulting double helix or with the first PNA strand.

We further have found that the PNA compounds are able to form triplehelices wherein a first PNA strand binds with the ssDNA or RNA or to oneof the strands of dsDNA and in doing so displaces the other strand, anda second PNA strand then binds with the resulting double helix. While wedo not wish to be bound by theory, it is further believed that othertriple helices might be formed wherein a single PNA strand binds to twosingle stranded nucleic acids strands. In binding with nucleic acidsboth Watson-Crick and Hoogsteen bind is utilized. It is further believedthat PNA might also bind via reverse Hoogsteen binding.

We have further surprisingly found that the PNA compounds form doublehelices with RNA and ssDNA. Such double helices are hetero duplexstructures between the PNA and the respective nucleic acid. Such doublehelices are preferably helices formed when the PNA strand includes amixture of both pyrimidine and purines nucleobases.

For therapeutic use of PNA compounds the targets of the PNA compoundswould generally be double stranded DNA and RNA. For diagnostic use,investigations methods and reagents where DNA is isolated outside of acell, the DNA can be denatured to single stranded DNA and use of the PNAcompound would be targeted to such single stranded DNA as well as RNA.

PNA compounds useful to effect binding to RNA, ssDNA and dsDNA and toform duplex and triplex complexes are in one sense polymeric strandsformed from a polyamide, polythioamide, polysulfinamide orpolysulfonamide backbone with a plurality of ligands located at spacedlocations along the backbone. At least some of the ligands are capableof hydrogen bonding with other ligands either on the compounds ornucleic acid ligands.

More preferred PNA compounds according to the invention have theformula: ##STR1## wherein: n is at least 2,

each of L¹ -L^(n) is independently selected from the group consisting ofhydrogen, hydroxy, (C₁ -C₄)alkanoyl, naturally occurring nucleobases,non-naturally occurring nucleobases, aromatic moieties, DNAintercalators, nucleobase-binding groups, heterocyclic moieties, andreporter ligands;

each of C¹ -C^(n) is (CR⁶ R⁷)_(y) where R⁶ is hydrogen and R⁷ isselected from the group consisting of the side chains of naturallyoccurring alpha amino acids, or R⁶ and R⁷ are independently selectedfrom the group consisting of hydrogen, (C₂ -C₆)alkyl, aryl, aralkyl,heteroaryl, hydroxy, (C₁ -C₆)alkoxy, (C₁ -C₆)alkylthio, NR³ R⁴ and SR⁵,where R³ and R⁴ are as defined above, and R⁵ is hydrogen, (C₁ -C₆)alkyl,hydroxy-, alkoxy-, or alkylthio- substituted (C₁ -C₆)alkyl, or R⁶ and R⁷taken together complete an alicyclic or heterocyclic system;

each of D¹ -D^(n) is (CR⁶ R⁷)_(z) where R⁶ and R⁷ are as defined above;

each of y and z is zero or an integer from 1 to 10, the sum y+z beinggreater than 2 but not more than 10;

each of G¹ -G^(n-1) is --NR³ CO--, --NR³ CS--, --NR³ SO-- or --NR³ SO₂--, in either orientation, where R³ is as defined above;

each of A¹ -A^(n) and B¹ -B^(n) are selected such that:

(a) A is a group of formula (IIa), (IIb), (IIc) or (IId), and B is N orR³ N⁺ ; or

(b) A is a group of formula (IId) and B is CH; ##STR2## where: X is O,S, Se, NR³, CH₂ or C(CH₃)₂ ;

Y is a single bond, O, S or NR⁴ ;

each of p and q is zero or an integer from 1 to 5, the sum p+q being notmore than 10;

each of r and s is zero or an integer from 1 to 5, the sum r+s being notmore than 10;

each R¹ and R² is independently selected from the group consisting ofhydrogen, (C₁ -C₄)alkyl which may be hydroxy- or alkoxy- oralkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen;and

each R³ and R⁴ is independently selected from the group consisting ofhydrogen, (C₁ -C₄)alkyl, hydroxy- or alkoxy- or alkylthio-substituted(C₁ -C₄)alkyl, hydroxy, alkoxy, alkylthio and amino;

Q is --CO₂ H, --CONR'R", --SO₃ H or --SO₂ NR'R" or an activatedderivative of --CO₂ H or --SO₃ H; and

I is --NHR'"R"" or --NR'"C(O)R"", where R', R", R'" and R"" areindependently selected from the group consisting of hydrogen, alkyl,amino protecting groups, reporter ligands, intercalators, chelators,peptides, proteins, carbohydrates, lipids, steroids, nucleosides,nucleotides, nucleotide diphosphates, nucleotide triphosphates,oligonucleotides, oligonucleosides and soluble and non-soluble polymers.

In the above structures wherein R', R", R'" and R"" are oligonucleotidesor oligonucleosides, such structures can be considered chimericstructures between PNA compounds and the oligonucleotide oroligonucleoside.

Preferred PNA-containing compounds useful to effect binding to RNA,ssDNA and dsDNA and to form triplexing structure are compounds of theformula III, IV or V: ##STR3## wherein: each L is independently selectedfromu the group consisting of hydrogen, phenyl, heterocyclic moieties,naturally occurring nucleobases, and non-naturally occurringnucleobases;

each R^(7') is independently selected from the group consisting ofhydrogen and the side chains of naturally occurring alpha amino acids;

n is an integer greater than 1, each k, l, and m is, independently, zeroor an integer from 1 to 5;

each p is zero or 1;

R^(h) is OH, NH₂ or --NHLysNH₂ ; and

R^(i) is H or COCH₃.

The improved binding of the PNA compounds of the invention withsingle-stranded RNA and DNA renders them useful as antisense agents. Inaddition, the binding to double-stranded DNA renders these compoundsuseful for gene inhibition via various mechanisms. Further, the bindingto double-stranded DNA renders these compounds useful as gene activatorsto initiate transcription.

In one embodiment, the present invention provides methods for inhibitingthe expression of particular genes in the cells of an organism,comprising administering to said organism a reagent as defined abovewhich binds specifically to sequences of said genes.

In a further embodiment, the invention provides methods for inhibitingtranscription and/or replication of particular genes or for modifyingdouble-stranded DNA as, for instance, by inducing degradation ofparticular regions of double-stranded DNA in cells of an organismcomprising administering to said organism a reagent as defined above.

In a still further embodiment, the invention provides methods forkilling cells or virus by contacting said cells or virus with a reagentas defined above which binds specifically to sequences of the genome ofsaid cells or virus.

A novel strategy for sequence-selective cleavage of double-stranded DNAis described. For cases were two closely positioned homo-pyrimidinestretches (of 7-10 bases and preferably on opposite strands) can beidentified, this can be done by synthesizing pairs of PNAs complementaryto two parts of this DNA sequence and separated by several base pairs.These PNA molecules are then reacted with the dsDNA and the resultingcomplex is allowed to react with an endonuclease.

In practicing certain embodiments of the invention, the PNA compoundsare able to recognize duplex DNA by displacing one strand, therebypresumably generating a hetero duplex with the other one. Suchrecognition can take place with dsDNA sequences 5-60 base pairs long.Sequences between 10 and 20 bases are of interest since this is therange within which unique DNA sequences of prokaryotes and eukaryotesare found. Reagents which recognize 17-18 bases are of particularinterest since this is the length of unique sequences in the humangenome.

The PNA compounds are able to form triple helices with dsDNA, ssDNA orRNA and double helices with RNA or ssDNA. In one embodiment of theinvention, the PNA compounds form triple helices wherein a first PNAstrand binds with a nucleic acid strand forming a hetero duplex and asecond PNA strand then binds with the resulting hetero duplex. In otherembodiments of the invention, a PNA compound or a PNA chimera compoundforms triple helices wherein a single PNA strand or PNA chimera strandbinds with two nucleic acid strands, with a nucleic acid strand and aPNA chimera strand or with two chimera PNA strands.

The invention further provides methods for inhibiting the action ofrestriction enzymes at restriction sites in nucleic acids. Such methodscomprise contacting a nucleic acid with a reagent as defined above underconditions effective to bind such reagent to the nucleic acid proximalto a restriction site.

The invention further provides methods of sequencing DNA by binding theDNA with a reagent as defined above at a site proximal to a restrictionsite, cleaving the DNA with a restriction enzyme, and identifying thecleaved products.

The invention further provides methods for initiating transcription incells or organisms comprising administering to the organism a reagent asdefined above which initiates transcription of a gene in such cells ororganisms.

The invention further provides methods for modulating binding of RNApolymerase to dsDNA by binding the dsDNA with a reagent as defined abovethat binds with the DNA and then exposing the complex formed thereby toa RNA polymerase.

The invention further provides methods for initiating transcription of agene by binding the gene with a reagent as defined above that interactswith the gene to melt the double-stranded DNA of the gene and to form atranscription elongation loop.

The invention further provides methods for binding RNA polymerase todsDNA by contacting the dsDNA with a reagent as defined above that iscapable of interacting with the DNA and then exposing the complex formedthereby to a RNA polymerase. More particularly, the interaction is viabinding to said dsDNA.

The invention further provides a hybrid complex for modulatingtranscription wherein the complex comprises dsDNA and a reagent asdefined above that binds with the dsDNA, and a RNA transferase.

The invention further provides a synthetic transcription factorcomprising a dsDNA and a reagent as defined above that is capable ofinteraction with the dsDNA. More particularly, the interaction is viabinding to said dsDNA.

The invention further provides specific gene activators comprising firstand second strands, as defined above, that have specific sequences thatbind to selected DNA regions of the gene.

The invention further provides chimeric structures comprising PNAs andDNA or RNA. Such chimeric structures will be used in place of or inaddition to a normal PNA strand to effect duplexing, triplexing, nucleicacid binding or protein binding.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an ethidium bromide stained gel showing cleavage in a sequencespecific manner by S₁ nuclease and a nucleic acid analogue of adouble-stranded DNA having a single site for hybridization to thenucleic acid analogue (Example 1);

FIG. 2 is an ethidium bromide stained gel showing a similar cleavage ofa double-stranded DNA having two closely spaced sites for hybridizationto the nucleic acid analogue on the same DNA strand (Example 2);

FIG. 3 is an ethidium bromide stained gel showing a similar cleavage ofa double-stranded DNA having two closely spaced sites for hybridizationto a nucleic acid analogue on opposite strands (Example 3).

FIG. 4 shows a PAGE autoradiograph demonstrating that PNAs-T₁₀, -T₉ Cand -T₈ C₂ bind to double-stranded DNA with high sequence specificy;

FIG. 5 shows an electrophoretic gel staining demonstrating thatrestriction enzyme activity towards DNA is inhibited when PNA is boundproximal to the restriction enzyme recognition site;

FIG. 6 shows a graph based on densitometric scanning of PAGEautoradiographs demonstrating the kinetics of the binding of PNA-T₁₀ toa double-stranded target;

FIG. 7 shows a graph based on densitometric scanning of PAGEautoradiographs demonstrating the thermal stabilities of PNAs of varyinglengths bound to an A₁₀ /T₁₀ double-stranded DNA target;

FIG. 8 is a schematic model of C⁺ G-G and T A-T triplets with Hoogsteenand Watson-Crick hydrogen bonds;

FIG. 9 is a schematic model of the binding of PNA (T₂ CT₂ CT₄) to adouble-stranded DNA target;

FIG. 10 shows a footprinting experiment of the binding of PNA to adimeric target;

FIG. 11 shows a footprinting experiment of PNA binding to a monomerictarget;

FIG. 12 is a footprinting experiment showing the effect of pH and salton the binding of a thymine/cytosine containing PNA to double strandedDNA;

FIG. 13 is an electron micrograph of a PNA double strand DNA complex;

FIG. 14 is a histogram representation of the results obtained in theexperiments of FIG. 13;

FIG. 15 is a one dimensional gel electrophoresis experiment showing theDNA unwinding upon PNA binding;

FIG. 16 is a two dimensional gel electrophoresis experiment showing theDNA unwinding upon PNA binding;

FIG. 17a shows the salt dependent binding of PNA in a gelelectrophoresis experiment;

FIG. 17b shows the salt resistance of the PNA-DNA complex in a gelelectrophoresis experiment;

FIG. 18 is an autoradiogram showing sequence selective transcriptioninhibition by PNA;

FIG. 19 is an autoradiogram showing sequence specific inhibition of TagDNA polymerase by PNA;

FIG. 20 is an autoradiogram showing the concentration dependence oftranscription inhibition by PNA T₁₀ ;

FIG. 21 is a FIG. 20 but using PNA T₄ CT₂ CT₂ ;

FIG. 22 is a quantitative representation of the results of FIGS. 20 &21;

FIG. 23 is an autoradiogram showing the results of transcription arrestusing various PNA and RNA polymerases;

FIG. 24 is a continuation of FIG. 23;

FIG. 25 is an autoradiogram showing the specific effect on transcriptionof PNA bound to the template versus the non-template strand;

FIG. 26 is an autoradiogram showing transcription arrest by short (T₆&T₈) PNAs;

FIG. 27 is a schematic drawing of PNA-DNA transcription initiationcomplexes;

FIG. 28 is an autoradiogram showing transcription initiation by PNA;

FIG. 29 is an autoradiogram showing the efficient competition of a "PNApromoter" versus the strong lacUV5 promoter;

FIG. 30 illustrates schematic structures of PNA/DNA complexes andsequences of the PNA targets;

FIG. 31 is a plasmid map;

FIG. 32 shows a PAGE autoradiography illustrating site specifictermination of in vitro transcription;

FIG. 33 shows a PAGE autoradiography the effect of target siteorientation on PNA mediated termination of in vitro transcription;

FIG. 34 shows a PAGE autoradiography illustrating the effect of saltconcentration on PNA activity;

FIG. 35 shows a PAGE autoradiography illustrating the effect of pH onPNA activity;

FIG. 36 are charts plotting K_(D) vs ph and K_(D) vs ionic strength onin vitro binding of PNA to complement;

FIG. 37 is a chart illustrating melting temperature for hybrid duplexbinding;

FIG. 38 is a chart illustrating melting temperature for hybrid duplexbinding;

FIG. 39 shows a PAGE autoradiography illustrating titration by gel-shiftbinding of PNA to a end labeled oligonucleotide;

FIG. 40 is a circular dichroism spectra;

FIG. 41 is a further circular dichroism spectra;

FIG. 42 is a chemical schematic; and

FIG. 43 is a further chemical schematic.

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of this invention the term "purine rich" shall mean apreponderance (i.e., greater than 50%) of purine bases, preferablygreater than about 65% and most preferrably greater than about 90%.Further, the term "modify" shall mean to change properties by additionto, substraction from, cleavage of or otherwise, such that which resultsis intrinsically different from that which is modified. The term"modulate" shall mean to change (i.e., increase or decrease) themagnitude of a property.

In accordance with the present inventions, the ligand L is primarily anaturally occurring nucleobase attached at the position found in nature,i.e., position 9 for purines including adenine, guanine or inosine, andposition 1 for yrimidines including thymine, uridine, or cytosine.lternatively, L may be a non-naturally occurring nucleobase (nucleobaseanalog), another base-binding moiety, an aromatic oiety, (C₁-C₄)alkanoyl, hydroxy or even hydrogen. It will be understood that theterm nucleobase includes nucleobases bearing removable protectinggroups. Some typical nucleobase ligands and illustrative syntheticligands are shown in FIG. 2 of WO 92/20702. Two particular nucleobaseligands, 5-propynylthymine and 3-deazauracil, have been shown toincrease binding affinity of an oligonucleotide to a target nucleotide(see Froehler, B. C. et. al., Tetrahedron Letters, 1992 33:5307-5310 andU.S. Pat. 5,134,066). Other like analogues are shown by Sagi, J. et.al., Tetrahedron Letters, 1993 34:2191-2194. Incorporation of thesenucleobase can be effected to increase the binding affinity of the PNAproduct with a nucleic acid target. Further useful non-naturallyoccurring nucleobases include 6-thioguanine, i.e., purine-6(1H)-thione,and pyrazolo[4,3-d]pyrimidines especially useful as triplexing bases(see PCT application PCT/US92/04795).

Furthermore, L can be a DNA intercalator, a reporter ligand such as, forexample, a fluorophor, a radio label, a spin label, hapten, or aprotein-recognizing ligand such as biotin. In monomer synthons, L may beblocked with protecting groups, as illustrated in FIG. 4 of WO 92/20702.

Linker A can be a wide variety of groups such as --CR¹ R² CO--, --CR¹ R²CS--, --CR¹ R² CSe--, --CR¹ R² CNHR³ --, --CR¹ R² C═CH₂ -- and --CR¹ R²C═C(CH₃)₂ --, where R¹, R² and R³ are as defined above. Preferably, A ismethylenecarbonyl (--CH₂ CO--), amido (--CONR³ --), or ureido (--NR³CONR³ --). Also, A can be a longer chain moiety such as propanoyl,butanoyl or pentanoyl, or corresponding derivative, wherein O isreplaced by another value of X or the chain is substituted with R¹ R² oris heterogenous, containing Y. Further, A can be a (C₂ -C₆)alkylenechain, a (C₂ -C₆)alkylene chain substituted with R¹ R² or can beheterogenous, containing Y. In certain cases, A can just be a singlebond.

In one preferred form of the invention, B is a nitrogen atom, therebypresenting the possibility of an achiral backbone. B can also be R³ N⁺,where R³ is as defined above, or CH.

In the preferred form of the invention, C is --CR⁶ R⁷ --, but can alsobe a two carbon unit, i.e., --CHR⁶ CHR⁷ -- or --CR⁶ R⁷ CH₂ --, where R⁶and R⁷ are as defined above. R⁶ and R⁷ also can be heteroaryl groupssuch as, for example, pyrrolyl, furyl, thienyl, imidazolyl, pyridyl,pyrimidinyl, indolyl, or can be taken together to complete an alicyclicsystem such as, for example, 1,2-cyclobutanediyl, 1,2-cyclopentanediylor 1,2-cyclohexanediyl.

In the preferred form of the invention, E in the monomer synthon is COOHor an activated derivative thereof, and G in the oligomer is --CONR³ --(amide). As defined above, E may also be CSOH, SOOH, SO₂ OH or anactivated derivative thereof, whereby G in the oligomer becomes --CSNR³--, --SONR³ --and --SO₂ NR³ --, (thioamide, sulfinamide or sulfonamide,respectively). The G group can be in either orientation, e.g., for amide--CONR₃ -- or --R₃ NCO--. The activation may, for example, be achievedusing an acid anhydride or an active ester derivative, wherein hydrogenin the groups represented by E is replaced by a leaving group suited forgenerating the growing backbone.

The amino acids which form the backbone may be identical or different.We have found that those based on 2-aminoethylglycine are especiallywell suited to the purpose of the invention.

In some cases it may be of interest to attach ligands at either terminus(Q, I) to modulate the binding characteristics of the PNAs.Representative ligands include DNA intercalators, which improve dsDNAbinding or basic groups, such as lysine or polylysine, which strengthenthe binding of the PNA due to electrostatic interaction. To decreaseelectrostatic repulsion charged groups such as carboxyl and sulfo groupscould be used. The design of the synthons further allows such othermoieties to be located on non-terminal positions. Oligonucleotidesand/or oligonucleoside can be covalently bound to terminal positions Qor I to form chimeras containing PNA portions and oligonucleotide and/oroligonucleoside portions. Nucleosides and/or nucleotides (mono, di ortri-phosphates) also can be attached to the terminal positions.

In a further aspect of the invention, the PNA oligomers are conjugatedto low molecular weight effector ligands such as ligands having nucleaseactivity or alkylating activity or reporter ligands (fluorescent, spinlabels, radioactive, protein recognition ligands, for example, biotin orhaptens). In a further aspect of the invention, the PNAs are conjugatedto peptides or proteins, where the peptides have signaling activity andthe proteins are, for example, enzymes, transcription factors orantibodies. Also, the PNAs can be attached to water-soluble orwater-insoluble polymers. In another aspect of the invention, the PNAsare conjugated to oligonucleotides or carbohydrates. When warranted, aPNA oligomer can be synthesized onto some moiety (e.g., a peptide chain,reporter, intercalator or other type of ligand-containing group)attached to a solid support.

Such conjugates can be used for gene modulation (e.g., gene targeteddrugs), for diagnostics, for biotechnology, and for scientific purposes.

As a further aspect of the invention, PNAs can be used to target RNA andssDNA to produce both antisense-type gene regulating moieties andhybridization probes for the identification and purification of nucleicacids. Furthermore, the PNAs can be modified in such a way that theyform triple helices with dsDNA. Reagents that bind sequence-specificallyto dsDNA have applications as gene targeted drugs. These are foreseen asextremely useful drugs for treating diseases like cancer, AIDS and othervirus infections, and may also prove effective for treatment of somegenetic diseases. Furthermore, these reagents may be used for researchand in diagnostics for detection and isolation of specific nucleicacids.

Triple helix formation, wherein an oligonucleotide is triplexed to adsDNA, is believed to be the only means known in the art forsequence-specific recognition of dsDNA. However, triple helix formationis largely limited to recognition of homopurine-homopyrimidinesequences. Triplexing with strand displacement using PNAs of thisinvention is superior to oligonucleotide-dsDNA triple helix recognitionin that it may allow for recognition of any sequence by use of the fournatural bases.

Gene targeted drugs are designed with a nucleobase sequence (containing10-20 units) complementary to the regulatory region (the promoter) ofthe target gene. Upon administration, the drug binds to the promoter andblocks access thereto by RNA polymerase. Consequently, no mRNA, and thusno gene product (protein), is produced. If the target is within a vitalgene for a virus, no viable virus particles will be produced.Alternatively, if the target is downstream from the promoter, RNApolymerase will terminate at this position, thus forming a truncatedmRNA/protein which is nonfunctional.

Sequence-specific recognition of ssDNA by base complementaryhybridization can be exploited to target specific genes and viruses. Inthis case, the target sequence is contained in the mRNA such thatbinding of the drug to the target hinders the action of ribosomes and,consequently, translation of the MRNA into protein. The peptide nucleicacids of the invention are superior to prior reagents in that they havesignificantly higher affinity for complementary ssDNA. Also while acharged species such as a lysine moiety can be added, PNAs possess nocharge, they are water soluble (which should facilitate cellularuptake), and they contain amides of non-biological amino acids (whichshould make them biostable and resistant to enzymatic degradation by,for example, proteases). Further, they can triplex with the mRNA.

The PNA compounds used in this invention can be synthesized by themethodologies disclosed in WO 92/20702, WO/92/20703 and the foregoingUnited States patent application bearing attorney docket ISIS-1017.Monomer synthons according to those disclosures are coupled usingstandard protocols to give the desired PNA oligomeric sequences. Thesynthesis of additional synthetic monomers are given by example 33through 45 of this specification.

Binding of PNA compounds to double-stranded DNA accompanied by stranddisplacement is shown in illustrative examples in this specification byboth enzymatic and chemical probing. Our illustrative examples show thatthe PNA compounds bind via Watson-Crick binding to their complementarystrand and extrudes the other strand in a virtually single-strandedconformation.

Using the PNA compound to locally unwind the DNA duplex and effectstrand displacement can render the displaced DNA strand sensitive tocleavage (e.g., cleavage by S₁ nuclease). This gives or induces highyield double strand DNA breaks at the site of PNA binding. PNA-directedand PNA-provoked double strand DNA cleavage by S₁ nuclease is especiallyefficient when two adjacent PNA sites are targeted, yieldingquanti-tative conversion of the target site into double-strand breaks.

As an illustrative example of use as an artificial restriction enzyme,the targets are cloned within a pUC19 polylinker and the plasmids arelinearized with the Cfr10I restriction enzyme. In the linear DNA, theresulting polylinker region is in the middle (1.33 kb from one end and1.36 kb from the other). A pT10 plasmid carrying a known sequence isthen inserted in the unique BamH1 site in the polylinker. This iscomplexed with a complementary PNA compound and subjected to treatmentwith S₁ nuclease. Upon nuclease treatment, a significant fraction of DNAis cut, as shown by gel electrophoresis of the resulting fragments.Cross reactivity experiments show that the targeting issequence-specific; only corresponding PNAs mediated cutting of thetargets. The yield of digested molecules is high and one can reach thequantitative digestion with increasing exposure to nuclease.

The results of such treatment demonstrate a novel method forsequence-selective cleavage of double-stranded DNA. If two closelypositioned homo-pyrimidine stretches can be identified (as for example7-10 bases, preferably on opposite strands), this can be done bysynthesizing pairs of PNAs complementary to two parts of this DNAsequence and separated by several base pairs. If this stranddisplacement binding mode is extended to include PNAs recognizing DNAsequences containing thymine and cytosine, this strategy allowstargeting and specific cleavage of any desired sequence of 10-20 basepairs. In effecting such cleavage, the high stability of PNA-DNAcomplexes consisting of two PNA strands (one "Watson-Crick" and one"Hoogsteen") and one DNA strand must be considered, as must the sequenceof the "Hoogsteen-like" strand.

We have found that complexes between homopyrimidine PNA andcomplementary oligonucleotides show 2:1 stoichiometry. Further we havefound that the thermal stability of triplexes betweencytosine-containing homopyrimidine PNA and complementaryoligonucleotides is strongly pH dependent, indicating the involvement ofC⁺ G-C Hoogsteen hydrogen bonding in (PNA)₂ /DNA triplexes. Further,while we do not wish to be bound by any particular theory, we believethat the strand displacement binding of homopyrimidine PNA to dsDNA isdependent on the formation of such (PNA)₂ /DNA triplexes with thecomplementary strand of the dsDNA target. It is our present belief thatformation of the strand displacement complex proceeds via a DNA/PNAWatson-Crick duplex which is subsequently stabilized by a Hoogsteenbound second strand having C⁺ -G Hoogsteen hydrogen bonds.

In further studies of triplexing and strand displacing, we utilized acytosine containing homopyrimidine PNA (T₂ CT₂ CT₄ -LysNH₂ ; SEQ IDNO:1) and studied its binding to a dimeric target. Stability of thecomplex was probed by both dimethylsulfate (DMS) probing and KMnO₄probing. In these studies, we found that triple strand formationinvolves Hoogsteen hydrogen bonding. This was evidenced by our showingthat the N7 atoms of guanines of the target participate in base pairingand, thus, are protected from reaction with DMS upon triplexing bindingof the PNA. A model of this triplexing binding is shown in FIGS. 8 and9.

We have also used electron microscopy to confirm strand displacement. Inthese examples, a PNA-DNA complex was formed with a target. Fulloccupancy of the target resulted in a strand displacement loop of 90-100bases, as detected by electron microscopy. The DNA molecules carry anopen region in the form of an "eye". In all cases, one of the twostrands in the open region is thicker than the other one and has thesame thickness as the normal DNA duplex. The thicker strand correspondsto the strand covered by PNA while the thinner strand corresponds to thedisplaced strand. In control experiments carried out without added PNA,the displaced structures are not observed.

Discrimination of PNA compounds in binding to targets was also observedutilizing labeled DNA fragments that were challenged with various PNAcompounds. We show that PNA compounds bind with high preference to thecomplementary target. Binding decreases with increasing mismatches inthe targets. Binding to a target containing one mismatch was weaker thanfor a fully complementary target, while no binding was observed with atwo-mismatch target.

We have further found that PNA compounds truly mimic DNA in terms ofbase pair specific hybridization to complementary strands ofoligonucleotides as measured by thermal stability of complexes. We haveshown that the formation of PNA/DNA duplexes exhibits a decrease inentropy almost identical to that for the formation of DNA/DNA duplexesand that like the formation of a DNA/DNA it is strongly enthalpy driven.From this we deduce that single stranded PNA must have the same degreeof base stacking as single stranded DNA and thus appears to be highlystructured. Further we have found that the rate of hybridization ofPNA/RNA duplexes is at least as fast as that for 2'-O-methyl RNA/RNA orDNA/DNA duplex formation again supporting our findings that singlestranded PNA is at least as prestructured for duplex formation as is DNAor RNA.

We have further found that in contrast to DNA or RNA, PNA compounds maybind to complementary DNA or RNA in one of two orientations, a parallelorientation or an anti-parallel orientation. We have further found thatthe PNA compounds prefer the anti-parallel orientation (wherein theamino end of the PNA strand is complementary to the 3'-end of thenucleic acid) over the parallel orientation ((wherein the amino end ofthe PNA strand is complementary to the 5'-end of the nucleic acid).

In our studies we have shown that homo-pyrimidine PNA binds strongly tossDNA, dsDNA or RNA with 2:1 stoichiometry (triplexing) and effectsstable strand displacement complexes with dsDNA. Further,purine-pyrimidine PNA binds to Watson-Crick complementaryoligonucleotides, either RNA or DNA, with 1:1 stoichiometry. Utilizingthese findings, one embodiment of this invention is directed to use ofPNA compounds as effective inhibitors of the elongation activity of RNAand DNA polymerases. As contrasted to oligonucleotides, this allows useof PNA compounds to target genes not only at promoter or regulatoryregions of the genome but to other regions as well. Furthermore, onlydecamer targets are required for efficient binding of the PNA to dsDNA.Consequently, it is easier to identify suitable PNA targets as comparedto oligonucleotide triple helix targets.

We have further found that PNA compounds have certain effects ontranscription in both prokaryotic and eukaryotic systems. Transcriptionby RNA polymerases T₃ or T₇ of PNA T₁₀ -LysNH₂ (SEQ ID NO:2) and T₂ CT₂CT₄ -LysNH₂ (SEQ ID NO:1) bound to dsDNA targets A₁₀ (SEQ ID NO:3), A₅GA₄ (SEQ ID NO:4) and A₂ GA₂ GA₄ (SEQ ID NO:5) positioned downstreamfrom T₃ or T₇ promoters in pBluescriptKS⁺ plasmids were studied. Asshown in our illustrative examples below, transcription elongation isarrested at the site of PNA binding to the template strand, whereas onlya marginal effect is observed at the site of PNA binding to thenon-template strand. With PNA T₁₀ -LysNH₂ (SEQ ID NO:2), transcriptionarrest occurs at the first base of the PNA binding site, while arrestwith PNA T₅ CT₄ -LysNH₂ (SEQ ID NO:6) takes place 2-3 bases inside thePNA binding site. In the case of PNA T₂ CT₂ CT₄ -LysNH₂ (SEQ ID NO:1),arrest is less efficient and occurs at the last 1-3 bases of the bindingsite.

Transcription arrest has also been shown for PNAs T₆ -LysNH₂ (SEQ IDNO:7) and T₈ -LysNH₂, although at lower efficiency compared to longerPNA compounds. These results show that efficient transcriptionelongation arrest can be obtained by PNA targeting of the transcribedstrand, and that "read through" by the polymerase takes place in asequence dependent manner.

Transcription arrest in eukaryotic systems has further been shown forPNA compounds. For these studies plasmids were constructed with the CMVIE 1 promoter driving the transcription of viral DNA sequences. Theviral DNA sequences containing homopurine target sites for the PNAs werecloned downstream of the CMV IE 1 promoter. The plasmids were incubatedwith PNA oligomers under various conditions then added to eukaryoticnuclear extracts to initiate in vitro transcription. Transcripts werespecifically truncated at the site of PNA binding. This effect wasdependent upon hybridization of the PNA to the template strand, i.e.,the inhibition of transcription was sequence specific. Binding affinitywas increased by either lowering the ionic strength or pH of the bindingbuffer. These results are consistent for PNA binding in whichhomopyrimidine PNA's invade duplex DNA binding the complementary DNAstrand, while displacing the non-complementary strand. A second PNA theninteracts with the PNA/DNA duplex forming a stable triple strandedstructure which is capable of blocking RNA polymerase II and terminatingtranscription thus specifically inhibiting eukaryotic gene expression.This use of PNA compounds to inhibit eukaryotic gene expression rendersthe PNA compounds useful for antigene therapeutics.

In a further embodiment of this invention, we have found that PNAcompounds also can be used as sequence specific gene activators andsynthetic transcription factors. Transcription initiation by RNApolymerase involves the sequence specific recognition of thedouble-stranded DNA promoter either by the polymerase itself or byauxiliary transcription factors. Subsequently a transcription initiationopen complex is formed in which about 12 base pairs of the DNA helix aremelted. This exposes the bases of the template strand for base pairingwith the RNA strand being synthesized. It has been shown that an E. coliphage T₇ RNA polymerase can utilize synthetic "RNA/DNA bubble duplex"complexes containing an RNA/DNA duplex and a single-stranded DNA D-loopfor transcription elongation. We have found that homopyrimidine PNAsalso form D-loop structures when binding to complimentarydouble-stranded DNA by strand displacement. We believe that thesestructures behave like RNA/DNA open complex structures and arerecognized by RNA polymerase.

In the illustrative examples below, we show that E. Coli RNA polymerasedoes indeed bind to PNA/dsDNA strand displacement complexes andinitiates RNA transcription therefrom. The results of these examplesfurther suggest that a single-stranded DNA loop is a major structuraldeterminant for RNA polymerase upon transcription initiation andelongation. The results of these examples also have implications forelucidating the mechanism of action of RNA polymerase.

In further embodiments of the invention chimera strands are formedbetween PNAs and either RNA or DNA. Such chimeric strands can then inthe same manner as the "homo" PNA strands described above. Such chimericstructures thus will be used, as described above, for binding,duplexing, triplexing and the like. In one particularly preferred use,either a PNA strand or a PNA containing chimera strand will be used tobind to or otherwise modulate proteins in cells. Such proteins willinclude transcription factors and other regulatory proteins.

The chimeric structure between PNAs and DNA or RNA are used in place ofor in addition to a normal PNA strand to effect duplexing, triplexing,nucleic acid binding or protein binding. The RNA or DNA nucleic acidportion of such chimeric structures include nucleic acid connected viaphosphodiester, phosphorothioate, phosphorodithioate, alkyl phosphonate,hydrogen phosphonate, phosphotriester, phosphoramidite and other likephosphorus linkages. They further can include other substitutions suchas substitution at the 2' position of a ribose sugar. Particularlypreferred are 2'-deoxy-2'-fluoro since they increase affinity of thenucleic acid portion of the chimera to other nucleic acids and2'-O-alkyl, particularly 2'-O-propyl, 2'-O-allyl and the like since theyconfer nuclease resistance to the nucleic acid strand.

The following examples are given to illustrate the invention. Theseexamples are given for illustrative purposes and are not meant to belimiting.

EXAMPLE 1

General Method for the Synthesis of PNA Oligomers

PNA oligomeric compounds were prepared generally in accordance with themethods disclosed by WO 92/20702, WO 92/20703 and the foregoing UnitedStates patent application bearing attorney docket ISIS-1017. Other PNAmonomers are prepared as per Examples 34-46 below. Briefly,benzyhydrylamine resin (initially loaded 0.28 mmol/gm withBoc-L-Lys(2-chlorobenzyloxycarbonyl)) was swollen in DMF and an excessof a monomer to be coupled was added, followed bydicyclohexylcarbodiimide (0.15M in 50% DMF in dichloromethane). The Bocdeprotection was accomplished by trifluoroacetic acid treatment. Theprogress of the coupling reactions was monitored by qualitative orquantitative ninhydrin analysis. The PNA was released from the resinusing anhydrous HF or trifluoromethyl sulfonic acid under standardconditions. The products were purified using HPLC withacetonitrile-water (0.1% TFA) gradient and structure confirmed by fastatom bombardment mass spectrometry.

Representative sequences synthesized by these methods include thefollowing as well as other sequences noted in the various examples:

H-T₁₀ LysNH₂ (SEQ ID NO:2)

H-T₄ CT₅ LysNH₂ (SEQ ID NO:8)

H-T₂ CT₂ CT₄ LysNH₂ (SEQ ID NO:1)

H-T₄ CT₂ CT₂ LysNH₂ (SEQ ID NO:9)

H-TGTACGTCACAACTA-NH₂ (SEQ ID NO:10)

H-CCTTCCCTT-NH₂ (SEQ ID NO:11l)

H-TTCCCTTCC-NH₂ (SEQ ID NO:12)

H-TAGTTATCTCTATCT-NH₂ (SEQ ID NO:13)

H-TGTACGTCACAACTA-NH₂ (SEQ ID NO:14)

H-GCACAGCC-LYS-NH₂ (SEQ ID NO:15)

H-TTTTCTTTT-NH₂ (SEQ ID NO:16)

H-TTTTTTTTTCCCCCCC-NH₂ (SEQ ID NO:17)

H-CCCCCCCTTTTTTTTT-NH₂ (SEQ ID NO:18)

H-CCTCCTTCCC-NH₂ (SEQ ID NO:19)

H-TTCTCTCTCT-NH₂ (SEQ ID NO:20)

H-TTTTTCTCTCTCTCT-NH₂ (SEQ ID NO:21)

H-CCCCCACCACTTCCCCTCTC-(Lys)₉ NH₂ (SEQ ID NO:22)

H-CTTATATTCCGTCATCGCTC-Lys-NH₂ (SEQ ID NO:23)

H-CTGTCTCCATCCTCTTCACT-NH₂ (SEQ ID NO:24)

H-TATTCCGTCATCGCTCCTCA-Lys-NH₂ (SEQ ID NO:25)

H-CCCCCACCACTTCCCCTCTC-NH₂ (SEQ ID NO:26)

H-CTGCTGCCTCTGTCTCAGGT-LysNH₂ (SEQ ID NO:27)

H-T₄ -(β-alanine)C-T₅ -LysNH₂ (SEQ ID NO:28)

H-T₄ -(β-alanine)T-T₅ -LysNH₂ (SEQ ID NO:29)

The PNA's are written from amino to the carboxyterminal. LysNH₂designates a lysine amide is attached to the PNA and NH₂ indicates afree c-terminal carboxamide without lysine.

EXAMPLE 2

Site-specific S₁ Nuclease Digestion of the pT10 Plasmid Linearized withCfrlOI Restriction Enzyme and Complexed with PNA (a Double-StrandedTarget)

PNA H-T₁₀ -LysNH₂ (SEQ ID NO:2) was synthesized as described inExample 1. A pT10 plasmid was prepared from the pUC19 plasmid byinserting the dA₁₀ /dT10 (SEQ ID NO:3) sequence into the BamH1 site ofthe polylinker as per the method of Egholm, M et al., J.A.C.S. 1992114:1895-1897. The pT10 plasmid was linearized with CfrlOI restrictionenzyme in the unique site. To form complex with the PNA, about 0.1 μg ofthe linearized plasmid was incubated with 2 o.u./ml of PNA in 3 μl ofthe TE buffer (10 mM Tris-HCI; 1 mM EDTA, pH 7.4) at 37° C. To performthe S₁ nuclease reaction, 10 μl of Na-Acetate buffer (33 mM NaAc; 50 mMNaCl; 10 mM ZnSO₄ ; 0.5% of glycerol, pH 4.6) and 145 units of S₁nuclease (Sigma) were added and incubated for various time periods atroom temperature. The reaction was terminated by adding 1 μl of 0.5MEDTA and cooling to -20° C. Electrophoresis was performed in 1% agarosegel in the TBE buffer with subsequent staining with ethidium bromide.The results of this example are shown in FIG. 1 wherein the size of themarkers are shown as are the incubation time periods. In FIG. 8 Lane 1:control--preincubation of DNA with PNA was performed under conditionsunfavorable for complex formation (200 mM of NaCl). Lane 2: thereference band obtained by digestion with DaHI restriction enzyme. Lane3: reference bands obtained by the digestion with Bgll restrictionenzyme. Lanes 4,5: the results of S₁ nuclease digestion for differenttimes of DNA-PNA complex.

As is shown in this example, targets were cloned within a pUC19polylinker. The plasmids were linearized with the CfrlOI restrictionenzyme. In the linear DNA, the obtained polylinker region is in themiddle (1.33 kb from one end and 1.36 kb from the other). Utilizing adirect approach, a pT10 plasmid carrying the sequence dA₁₀ dT₁₀ (SEQ IDNO:31) was inserted in the unique BamH1 site in the polyIinker. This wascomplexed with PNA H-T₁₀ -LysNH₂ (SEQ ID NO:2) and subjected totreatment with S₁ nuclease. After treatment with 145 units of S₁nuclease at room temperature a significant fraction of DNA was cut. Themobility by gel electrophoresis of the resulting fragments was veryclose to the mobility of fragments obtained by cutting with restrictionenzyme BamH1. Similar results were obtained for H-T₅ CT₄ -LysNH₂ (SEQ IDNO:6) and H-T₂ CT₂ CT-LysNH₂ (SEQ ID NO:51) PNAs and correspondingplasmids carrying the d(A₅ GA₄)/d(T₄ CT₅) (SEQ ID NO:4) and d(A₄ GA₂GA₂)/d(T₂ CT₂ CT4) (SEQ ID NO:44) inserts. Cross reactivity experimentsshowed that the targeting was sequence specific: only corresponding PNAsmediated cutting of the targets. It was noted that two very week bandsare seen in lanes 4,5, which correspond to lengths 2.32 and 0.98 kb.These bands are due to weak binding of PNA-TIO to the intrinsic pUC19site d(TTGT3)/d(A3CAT), which is 0.37 kb apart from the Cfr10Irestriction site. This is consistent with the results of Example 3 abovethat shows that the introduction of mismatches dramatically decreasesthe affinity of PNA to DNA.

From this example it is believed that the enzyme first digests thedisplaced strand, then to some extent enlarges the gap after which theopposite strand becomes a substrate for the enzyme. As a result, thedouble-stranded break is created. The yield of digested molecules ishigh and one can reach the quantitative digestion with increasingexposure to S₁ nuclease (data not shown). However, while the twofragments are poorly resolved after digestion with BamHI restrictionenzyme, the PNA-mediated digestion with the S₁ nuclease leads to theclear-cut doublet. While not wishing to be bound by theory, we believethis probably reflects a widening of the gap by the S₁ nuclease as wellas digestion from the ends of the cfr10I site. With longer treatment thedownward shift of the doublet becomes noticeable (see lane 5). Thus,quantitative digestion is clearly accompanied by a truncation of thefragments (by about hundred base pairs). To obtain further precision ofcutting comparable with that exhibited by natural restriction enzymesthe original approach is modified as per Example 4 below.

Alternate site-specific S₁ nuclease digestion of plasmid with PNA)

As an alternative target, the insert:

5'-A₅ GA₄ GTCGACA₅ GA₄ -3' (SEQ ID NO:30)

3'-T₅ CT₄ CAGCTGT₅ CT₄ -5'

was cloned into the Sall site of the pUC19 plasmid. This plasmid, whichcontained two binding sites for PNA H-T₅ CT₄ -LysNH₂ (SEQ ID NO:6)separated by six base pairs, was designated pT9CT9C. Strand displacementin the two T₅ CT₄ sites led to opening of the entire region, includingthe sequence GTCGAC/GACGAC, providing a substrate for the S₁ nuclease inboth strands. As control, a pT9C-5 plasmid was used, which carried onlyone T₅ CT4 insert cloned into the Sall site of the PUC19 plasmid.

EXAMPLE 3

Site-specific S₁ Nuclease Digestion of the pT9C Plasmid Linearized byCfr10I Restriction Enzyme and Complexed with PNA H-T₅ CT₄ -LysNH₂

This example illustrates the use of one molecule of PNA hybridized to atarget DNA to define a restriction site.

PNA 1,2 and 3 were synthesized as Example 1 and the results are shown inFIG. 2. A pT9C plasmid carried the insert:

5'-A₅ GA₄ GTCGACA₅ GA₄ -3' (SEQ ID NO:30)

3'-T₅ CT₄ CAGCTGT₅ CT₄ -5',

cloned in the Sal1 site of the pUC19 polylinker. The pT9C-5 plasmidcarried the single insert A₅ GA₄ /CT₅ cloned in the same site. ThePNA-DNA complexes were prepared as described in Example 2 with the onlydifference being the duration of the incubation was 2 hours. Digestionby 30 units of the S₁ nuclease was performed in the same buffer asdescribed in Example 2 with two exceptions, lanes 2 and 4. In lane 2, 15units of the enzyme were used, whereas in lane 4 200 mM NaCl and 1 mM ofZnSO₄ were added to the buffer. Lane 1: the reference band obtained bydigestion with the BamHI restriction enzyme of the pUC19 plasmidlinearized by the Cfr10I restriction enzyme. Lanes 2-4 site-specificdigestion by the S₁ nuclease of the linearized pT9C plasmid complexedwith PNA H-T₅ CT₄ -LysNH₂ (SEQ ID NO:6). Lanes 5-7: various controls.Lane 8: the same experiment as in lane 3, but using PNA T₁₀ (SEQ IDNO:2) instead of T₄ CT₅ (SEQ ID NO:8). Lane 9: the same experiment as inlane 3, but using PNA T₂ CT₂ CT₄ (SEQ ID NO:1) instead of T₄ CT₅ (SEQ IDNO:8). Lane 10: the same experiment as in lane 3, but using the pT9C-5plasmid instead of the pT9C. In the header PNA T₅ CT₄ (SEQ ID NO:6) islabelled as 1, T₁₀ (SEQ ID NO:2) as 2 and T₂ CT₂ CT₄ (SEQ ID NO:1) as 3.

This example show that subjecting the pT9CT9C plasmid, linearized by theCfr10I restriction enzyme and complexed with PNA H-T₅ CT₄ -LysNH₂ (SEQID NO:6), to 30 units of the S₁ nuclease results in full conversion ofthe full-length DNA molecules into half-length fragments. The bandsobserved by gel electrophoresis have the same position and width as theband created by the BamHI restriction enzyme. Sequence specificity ofthis artificial "restriction enzyme" was confirmed by the very weakdigestion of the pT9C plasmid in case of complexing with PNA H-T₁₀-LysNH₂ (SEQ ID NO:2) and H-T₂ CT₂ CT₄ -LysNH₂ (SEQ ID NO:1) underconditions which result in quantitative cutting of the plasmid in thepresence of PNA H-T₅ CT₄ -LysNH2 (SEQ ID NO:6) (see lanes 8,9).Moreover, under the much milder S₁ nuclease treatment necessary togenerate the data for the pT9CT9C, the yield of double-stranded breaksin the pT9C-5 plasmid was extremely low (lane 10).

EXAMPLE 4

Site Specific S₁ Nuclease Cleavage of the Plasmids pT9C-5, pT9CT9C andpT9CA9GKS (Linearized with Sca1) Targeted by PNA T₄ CT₅ -LysNH₂

This example illustrates the use of two molecules of PNA bound toopposite strands of a DNA target. A further plasmid pT9CA9GKS wasproduced by cloning the insert GTCGACA₅ GA₄ GTCGACT₄ CT₅ GTCGAC (SEQ IDNO:32) into pUC19 at the Sal I site and linearizing with Sca Irestriction enzyme. The linearized plasmid has two hybridization sitesfor PNA H-T₄ CT₅ -Lys NH₂ (SEQ ID NO:8) on opposite strands spaced bysix base pairs. The protocol of Example 3 was use except the sampleswere treated using 1 U/μl of S₁ and 15 min incubation at 37° C. Theresults are shown in FIG. 3 Lanes 1-4: pT9C-5; lanes 5-8: pT9CT9C; lanes9-12: pT9CA9GKS. Lanes 1, 5 & 9: no PNA; lanes 2, 6 & 10: 50 μM; lanes3, 7, 11: 500 uM; lanes 4, 8, 12: 5 mM.

EXAMPLE 5

Use of PNA/Nuclease to Cut Selectively Large DNA Molecule

The standard strain lambda cI indl ts857 Sam7 (New England Biolab) wasused. The PNA double target was T₇ N₆ T₇ (SEQ ID NO:33) which uponcleavage should give rise to a 6.1 kb fragment. The PNA was a PNA (T₇)₂and the conditions were as follows: 0.1 μg DNA and 1-2 OD units of PNAwere incubated in 5 μl TE buffer for 10 min at 37° C. 5 μl buffer (33 mMNaAc, 30 mM NaCl, 10 μM ZnSO₄, pH 4.5) and 20 U mung bean nuclease wereadded and the mixture was incubated for 5 min at room temperature. Uponanalysis by electrophoresis in 0.5% agarose in TBE buffer, fragments ofsizes 6 kb and 42 kb were observed using lambda×Hind III or Sal I assize markers. Without additions of PNA no bands were seen.

This example demonstrates the ability of PNA/nuclease to cut selectivelya large DNA molecule (by comparison to the much smaller plasmids used inExamples 1 to 3).

EXAMPLE 6

Binding of PNAs-T₁₀ /T₉ C/T₈ C₂ to Double-stranded DNA Targets A₁₀ /A₉G/A₈ G₂

A mixture of 200 cps ³² P-labeled EcoRI-PvuII fragment (the largefragment labeled at the 3'-end of the EcoRI site) of the indicatedplasmid, 0.5 μg carrier calf thymus DNA, and 300 ng PNA in 100 μl buffer(200 mM NaCl, 50 mM Na-acetate, pH 4.5, 1 mM ZnSO₄) was incubated at 37°C. for 120 min. A 50 unit portion of nuclease S₁ was added and incubatedat 20° C. for 5 min. The reaction was stopped by addition of 3 μl 0.5MEDTA and the DNA was precipitated by addition of 250 μl 2% potassiumacetate in ethanol. The DNA was analyzed by electrophoresis in 10%polyacrylamide sequencing gels and the radiolabeled DNA bands visualizedby autoradiography.

The target plasmids were prepared by cloning of the appropriateoligonucleotides into pUC19. Target A₁₀ : oligonucleotides GATCCA₁₀ G(SEQ ID NO:34) & GATCCT₁₀ G (SEQ ID NO:35) cloned into the BamHI site(plasmid designated pT10). Target A₅ GA4: oligonucleotides TCGACT₄ CT₅ G(SEQ ID NO:36) & TCGACA₅ GA₄ G (SEQ ID NO:37) cloned into the SalI site(plasmid pT9C). Target A₂ GA₂ GA₄ : oligonucleotides CA₂ GA₂ GA₄ CTGCA(SEQ ID NO:38) and GT₄ CT₂ CT₂ CTGCA (SEQ ID NO:39) into the PstI site(plasmid pT8C2). The results are shown in FIG. 4. The positions of thetargets in the gel are indicated by bars to the left. A/G is an A+Gsequence ladder of target P10.

EXAMPLE 7

Inhibition of Restriction Enzyme Cleavage by PNA (FIG. 4)

A 2 μg portion of plasmid pT10 was mixed with the indicated amount ofPNA-T₁₀ (SEQ ID NO:2) in 20 μl TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH7.4) and incubated at 37° C. for 120 min. 2 μl 10× buffer (10 mMTris-HCl, pH 7.5, 10 MM, MgCl₂, 50 mM NaCl, 1 mM DTT). PvuII (2 units)and BamHI (2 units) were added and the incubation was continued for 60min. The DNA was analyzed by gel electrophoresis in 5% polyacrylamideand the DNA was visualized by ethidium bromide staining. This gel isillustrated in FIG. 5.

EXAMPLE 8

Kinetics of PNA-T₁₀ --dsDNA Strand Displacement Complex Formation

A mixture of 200 cps ³² P-labeled EcoRI-PvuII fragment of pT10 (thelarge fragment labeled at the 3'-end of the EcoRI site), 0.5 μg carriercalf thymus DNA, and 300 ng of PNA-T₁₀ -LysNH₂ (SEQ ID NO:2) in 100 μlbuffer (200 mM NaCl, 50 mM Na-acetate, pH 4.5, 1 mM ZnSO₄) wereincubated at 37° C. At the times indicated, 50 U of S₁ nuclease wasadded to each of 7 samples and incubation was continued for 5 min at 20°C. The DNA was then precipitated by addition of 250 μl 2% K-acetate inethanol and analyzed by electrophoresis in a 10% polyacrylamidesequencing gel. The amount of strand displacement complex was calculatedfrom the intensity of the S₁ -cleavage at the target sequence, asmeasured by densitometric scanning of autoradiographs. The results areillustrated in FIG. 6.

EXAMPLE 9

Stability of PNA-dsDNA Complexes

A mixture of 200 cps ³² P-pT10 fragment, 0.5 μg calf thymus DNA and 300ng of the desired PNA (either T₁₀ -LysNH₂ (SEQ ID NO:2), T₈ -LysNH₂ (SEQID NO:40) or T₆ -LysNHz (SEQ ID NO:7)) was incubated in 100 μl 200 mMNaCl, 50 mM Na-acetate, pH 4.5, 1 mM ZnSO₄ for 60 min at 37° C. A 2 μgportion of oligonucleotide GATCCA₁₀ G (SEQ ID NO:34) was added and eachsample was heated for 10 min at the temperature indicated, cooled in icefor 10 min and warmed to 20° C. A 50 U portion of S₁ nuclease was addedand the samples treated and analyzed and the results quantified as isillustrated in FIG. 7.

EXAMPLE 10

Biological Stability of PNA

A mixture of PNA-T₅ (SEQ ID NO:41) (10 μg) and a control, "normal"peptide (10 μg) in 40 μl 50 mM Tris-HCl, pH 7.4 was treated with varyingamounts of peptidase from porcine intestinal mucosa or protease fromStreptomyces caespitosus for 10 min at 37° C. The amount of PNA andpeptide was determined by HPLC analysis (reversed phase C-18 column:0-60% acetonitrile, 0.1% trifluoroacetic acid).

At peptidase/protease concentrations where complete degradation of thepeptide was observed (no HPLC peak) the PNA was still intact.

EXAMPLE 11

Inhibition of Gene Expression

A preferred assay to test the ability of peptide nucleic acids toinhibit expression of the E2 MRNA of papillomavirus is based on thewell-documented transactivation properties of E2. Spalholtz, et al., J.Virol., 1987, 61, 2128-2137. A reporter plasmid (E2RECAT) wasconstructed to contain the E2 responsive element, which functions as anE2 dependent enhancer. E2RECAT also contains the SV40 early promoter, anearly polyadenylation signal, and the chloramphenicol acetyl transferasegene (CAT). Within the context of this plasmid, CAT expression isdependent upon expression of E2. The dependence of CAT expression on thepresence of E2 has been tested by transfection of this plasmid into C127cells transformed by BPV-1, uninfected C127 cells and C127 cellscotransfected with E2RECAT and an E2 expression vector.

A. Inhibition of BPV-1 E2 Expression

BPV-1 transformed C127 cells are plated in 12 well plates. Twenty fourhours prior to transfection with E2RE1, cells are pretreated by additionof antisense PNAs to the growth medium at final concentrations of 5, 15and 30 mM. The next day cells are transfected with 10 μg of E2RE1CAT bycalcium phosphate precipitation. Ten micrograms of E2RE1CAT and 10 μg ofcarrier DNA (PUC 19) are mixed with 62 μl of 2M CaCl₂ in a final volumeof 250 μl of H₂ O, followed by addition of 250 μl of 2× HBSP (1.5 mM Na₂PO₂. 10 mM KCl, 280 mM NaCl, 12 mM glucose and 50 mM HEPES, pH 7.0) andincubated at room temperature for 30 minutes. One hundred microliters ofthis solution is added to each test well and allowed to incubate for 4hours at 37° C. After incubation, cells are glycerol shocked for 1minute at room temperature with 15% glycerol in 0.75 mM Na₂ PO₂, 5 mMKCl, 140 mM NaCl, 6 mM glucose and 25 mM HEPES, pH 7.0. After shocking,cells are washed 2 times with serum free DMEM and fed with DMEMcontaining 10% fetal bovine serum and antisense oligonucleotide at theoriginal concentration. Forty eight hours after transfection cells areharvested and assayed for CAT activity.

For determination of CAT activity, cells are washed 2 times withphosphate buffered saline and collected by scraping. Cells areresuspended in 100 μl of 250 mM Tris-HCl, pH 8.0 and disrupted byfreeze-thawing 3 times. Twenty four microliters of cell extract is usedfor each assay. For each assay the following are mixed together in an1.5 ml Eppendorf tube and incubated at 37° C. for one hour: 25 μl ofcell extract, 5 μl of 4 mM acetyl coenzyme A, 18 μl H₂ O and 1 μl ¹⁴C-chloramphenicol, 40-60 mCi/mM. After incubation, chloramphenicol(acetylated and nonacetylated forms) is extracted with ethyl acetate andevaporated to dryness. Samples are resuspended in 25 μl of ethylacetate, spotted onto a TLC plate and chromatographed inchloroform:methanol (19:1). Chromatographs are analyzed byautoradiography. Spots corresponding to acetylated and nonacetylated ¹⁴C-chloramphenicol are excised from the TLC plate and counted by liquidscintillation for quantitation of CAT activity. Peptide nucleic acidsthat depress CAT activity in a dose dependent fashion are consideredpositives.

B. Inhibition of HPV E2 Expression

The assay for inhibition of human papillomavirus (HPV) E2 by peptidenucleic acids is essentially the same as that for BPV-1 E2. For HPVassays appropriate HPVs are co-transfected into either CV-1 or A431cells with PSV2NEO using the calcium phosphate method described above.Cells which take up DNA are selected for by culturing in mediacontaining the antibiotic G418. G418-resistant cells are then analyzedfor HPV DNA and RNA. Cells expressing E2 are used as target cells forantisense studies. For each PNA, cells are pretreated as above,transfected with E2RE1CAT, and analyzed for CAT activity as above.Peptide nucleic acids are considered to have a positive effect if theycan depress CAT activity in a dose dependent fashion.

EXAMPLE 12

Triplexing of PNA to Nucleic acids--Probing Protocols

Probing was effected in 100 μ buffer (S1: 100 mM NaCl, 1 mM ZnSO₄, 50 mMNaAc, pH 4.5; KMnO₄ /dimethyl sulphate (DMS): 10 mM Na-cacodylate, 1 mMEDTA, pH 7.0 or as other wise noted) containing about 200 cps ³²P-labeled DNA fragment, 0.5 μg calf thymus DNA and the desired amount ofPNA. Following a preincubation for 60 min at 37° C., the probing reagentwas added and the incubation was continued at room temperature. Thereactions were terminated by the addition of a stop-buffer. The DNA wasprecipitated by addition of 200 μL 2% KAc in 96% EtOH and was analyzedby electrophoresis in 10% polyacrylamide sequencing gels. RadioactiveDNA bands were visualized by autoradiography using amplifying screensand Agfa curix RPA X-ray films exposed at -70° C.

Probing conditions were: S1: 0.5 U/μl, 5 min. stopped with 3 μl 1M EDTA;KMnO₄ : 1 mM, 15 sec. stopped with 50 μl 1M 6-mercaptoethanol, 1.5MNaAc, pH 7.0; DMS: 1% DMS, 15 sec., stopped as for KMnO₄ probing.Samples probed with DMS or KMnO₄ were treated with piperidine (0.5M, 90°C., 20 min.) prior to gel analysis.

EXAMPLE 13

Enzymatic and Chemical Probing of the Binding of PNA T₂ CT₂ CT₄ -LysNH₂to pT8C2A8G2

For this example the 264 bp EcorI/PvuII fragment of pT8C2A8G2 that was3'-³² P-end-labeled at the EcorI site was used. Probing was effected asper the protocols of Example 16. The results are shown in FIG. 10. Lane1 is a control in S1 buffer without S₁ nuclease and without PNA. Lanes2-5: S1 probing, lanes 6-9: KMnO₄ probing and lanes 10-13: DMS probing.The following concentrations of PNA were used: 0 μM (lanes 2, 6 & 10),0.25 μM (lanes 3, 7 & 11), 2.5 μM (lanes 4, 8 & 12) or 25 μM (lanes 5, 9& 13). Lanes S are A+G sequence markers.

The plasmid pT8C2A8G2 contains two targets for PNA T₂ CT₂ CT₄ (SEQ IDNO:1) in opposite orientation. Therefore both KMnO₄ (thymines of thepyrimidine strand) and DMS (guanines of the purine strand) probing wasdone on the same DNA fragment. Probing with single strand specificnuclease, S₁, verified that upon binding of PNA, the pyrimidine strandbut not the purine strand of the PNA targets, became susceptible toattack by S₁ (FIG. 10, lanes 2-5). Interestingly, however, in theabsence of PNA S₁ attacks the purine strand (lane 2). This isundoubtedly due to the formation of an intramolecular triple helix(H-DNA), a well characterized feature of such polypurine/pyrimidinemirror repeats. It is also observed that in the presence of PNA, the SIsensitivity extends into the linker between the two PNA targets. Thiscan be explained if both PNA targets are occupied, thereby forming acombined loop where the DNA region between the targets issingle-stranded. This type of binding further illustrates that PNA canbe used for directed double strand DNA cleavage by S₁.

The KMnO₄ probing of the PNA-dsDNA complex reveals that all thymines ofthe target are oxidized by KMnO₄ (FIG. 13, lanes 8-9). Concomitantly (interms of PNA concentration) with the occurrence of KMnO₄ susceptibility,virtually full protection against reaction with DMS is observed at thetwo guanines of the opposite PNA target (see FIG. 10, lanes 12-13).These results show that displacement of the pyrimidine strand of the DNAtarget is accompanied by protection of the major groove upon binding ofPNA supporting the model in which two PNA strands are participating inthe PNA-DNA complex forming PNA DNA-DNA triplex.

EXAMPLE 14

Chemical Probing of the Binding of PNA T₂ CT₂ CT₄ -LysNH₂ to pA8G2

For this example, the 248 bp EcoRI/PvuII fragment of pA8G2 was used thatwas ³² P-end-labeled (5'-labeling, lanes 1-5) or (3'-labeling, lanes6-9)) at the EcoRI site. Probing was effected as per the protocols ofExample 12. The results are shown in FIG. 11 wherein lane 1 is a controlin S₁ buffer without S₁ nuclease and without PNA. Lanes 2-5: KMnO₄probing and lanes 6-9: DMS probing. The following concentrations of PNAwere used: 0 μM (lanes 2 & 6), 0.25 μM (lanes 3 & 7), 2.5 μM (lanes 4 &8) or 25 μM (lanes 5 & 9). Lanes S are A+G sequence markers.

In this example, in order to assure that the results were not anartifact of the dimeric target, we performed a similar experiment tothat of Example 13 except a plasmid (pA8G2) having only a single PNAtarget was used. As is seen in FIG. 14, virtually identical results tothose with the dimeric target of Example 13 were obtained with KMnO₄ andDMS probing of the single target.

EXAMPLE 15

Effect of pH and Ionic Strength of the Binding of PNA T₂ CT₂ CT₄ -LySNH₂to pTSC2A8G2

For this example the 264 bp EcorI/PvuII fragment of pT8C2A8G2 was usedthat was 3'-³² P-end-labeled at the EcorI site. Probing was effected asper the protocols of Example 12. The results are shown in FIG. 12wherein lanes 1 & 9 are controls without PNA. Lanes 1-7 are KMnO₄probing while lanes 9-15 are DMS probing. The samples of lanes 2-7 &9-15 contained 5 μM PNA. The samples of lanes 3, 5, 7, 11, 13 & 15contained 100 mM NaCl in the 10 mM Na phosphate, 1 mM EDTA buffer. ThepH of the buffer was 7.5 (lanes 2, 3 & 10, 11); 6.5 (lanes 4, 5 & 12,13) or 5.5 (lanes 6,7 & 14, 15). Lanes are A+G sequence markers.

This example shows that the Hoogsteen hydrogen bonding of cytosine toguanine requires protonation of N3 of cytosine and consequentlyinteractions involving C⁺ -G Hoogsteen base pairing are very sensitiveto the acidity of the medium. This has been studied extensively byothers with DNA triple helices, and can also be seen with (PNA)₂ /DNAtriplexes using oligonucleotides.

The results presented in FIG. 12 further show that the stranddisplacement binding of PNA T₂ CT₂ CT₄ (SEQ ID NO:1) to the target ofpT8C2A8G2 is also sensitive to pH. Very little binding is observed at pH7.5 (lanes 2 & 10) compared to pH 6.5 (lanes 4 & 12) and 5.5 (lanes 6 &14). Full consistency is also seen between the KMnO₄ and DMS probingresults showing clear correlation between strand displacement andHoogsteen type binding of the PNA. We have previously shown that mediumionic strength (50-100 mM Na⁺) inhibits strand displacement binding ofPNA to double-stranded DNA. The results presented in FIG. 15 (lanes 3,5, 7) are fully consistent with this. There is no reason fromphysio-chemical considerations that binding of PNA to DNA as aconventional PNA DNA-DNA triple helix should be strongly salt dependent.Therefore, such complexes could be envisaged under salt conditions thatdisfavor strand displacement. However, we see no evidence that this bethe case since no DMS footprint is observed under "high-salt" conditions(FIG. 12, lanes 11, 13 & 15).

From Examples 13, 14 and 15 as well as other of the Examples of thisspecification, we conclude that sequence specific binding of PNA todouble-stranded DNA involves PNA DNA-DNA triplexes employingconventional Hoogsteen and Watson-Crick base pair hydrogen bonding forrecognition.

EXAMPLE 16

Electron Microscopy A pA98 plasmid containing an A₉₈ /T₉₈ (SEQ ID NO:42)insert in the PstI site of the polylinker of a pUC19 plasmid was used.0.1 μg of the pA98 plasmid linearized by the Cfr10I I restriction enzymewas incubated with 0.15 μg of PNA H-T₁₀ -LysNH₂ at 37° C. for 3 hours in10 μL of the TE buffer (10 MM of Tris-HCl, 1 mM of Na₃ EDTA, pH 8.0).The complex was diluted with buffer containing 10 mM of Tris-HCI, 10 mMof NaCl, pH 7.5 to the final DNA concentration of 0.2-0.5 μg/mL andabsorbed to the surface of glow discharged in tripropylamine carbon gridfor 2 minutes as described in Duochet, J., Ultrastruct. Res., 1971 35,147-167. The grid was stained in 0.1-0.5% water solution of uranylacetate for 10-15 sec and dried. The sample was shadowed with Pt/C(95/5) and studied in a Philips 400 electron microscope at acceleratingvoltage of 40 kV. The length of the DNA molecules were measured and ahistogram was plotted as described by A. V. Kurakin in MicronMicroscopica Acta, 22,213-221 (1991). The length of unwound region wasmeasured by its thicker strand, for which the number of base pairs perunit length was assumed to be the same as for the DNA duplex. Foranalysis by use of a histogram, the molecules were oriented in such amanner that the left boundary of the loop was closer to the end of themolecule.

The electron microscopy of the PNA-DNA complex was carried out on theA₉₈ /T₉₈ (SEQ ID NO:42) target contained within the supercoiled plasmid,pA98, linearized with the Cfr10I restriction enzyme, and challenged witha PNA H-T₁₀ -LysNH₂ (SEQ ID NO:2). Full occupancy of the target resultsin a strand displacement loop of 90-100 bases was detected as is shownin FIG. 13. This histogram plot is illustrated in FIG. 14. As is seen inFIG. 13, the DNA molecules carry an open region in the form of "an eye".In all cases one of the two strands in the open region was thicker thanthe other one and has the same thickness as the normal DNA duplex. Thethicker strand corresponds to the A-strand covered by PNA, while thethinner strand corresponds to the displaced T-strand. The positions oftwo branch points of the loops were 47.1% and 51.2%, which within theerror of EM-measurements (0.5%), coincides with the ends of the A98/T98insert (47.5% and 51.0%). The average size of the loop was 4.1%, i.e.,114 bp, which also agrees well with the length of the insert (thestandard error is 17bp). In control experiments carried out withoutadded PNA, the eye-like structures were not observed.

EXAMPLE 17

Unwinding of Closed Circular DNA with PNA

Relaxed circular DNA was prepared by treating ordinary supercoiledplasmid DNA with an extract containing DNA topoisomerase I, as describedby V. I. Lyamichev, et. al., J. Biomolec. Struct. Dynamics, 3, 327-338(1985). DNA-PNA complexes were obtained by incubation of 1-2 μg of DNAin 5-20 μL TE with 0.4 optical units./mL of PNA for 4 hours at 20-22° C.This corresponded to about 10 times molar excess of PNA to its potentialbinding sites. Agarose (1.5%) gel electrophoresis was performed in theTAE buffer containing 1 μg/mL of chloroquine at 10° C. for 15 hours at1.5 V/cm. Two-dimensional gel electrophoresis was preformed in the firstdirection (from top to bottom) in the TAE buffer and in the seconddirection (from left to right) in the same buffer with addition of 1μg/mL of chloroquine.

In this example, the efficiency of PNA T10 incorporating into the DNAduplex and displacing the DNA T-strand in solution was shown byunwinding of closed circular DNA by PNA. The pA98 plasmid (Example 16)was prepared in the form of relaxed circles (rcDNA). It was expectedthat complexing with PNA would unwind about 10 turns of the DNA duplex.Because of topological constraints (the two strands in rcDNA were closedand therefore topologically linked), this unwinding would make the rcDNAmolecules behave as if they were positively supercoiled by 10superturns. This would manifest itself in an increase of electrophoreticmobility of the complex as compared with control rcDNA preincubated inthe same buffer without PNA. This increase in electrophoretic mobilityis shown in FIG. 15 that shows that after incubation with PNA in thelow-salt TE buffer for 4 hours at 20° C., virtually all rcDNA moleculessignificantly increased their mobility and moved as if they were highlysupercoiled. Two-dimensional gel electrophoresis gels, FIG. 16, showsthat these molecules behave actually as positively supercoiled ones.This technique is used to resolve individual topoisomers. Using a samplewith a wide distribution of topoisomers as reference, it is clearly seenFIG. 16 that on the average 8-10 turns of the double helix are releasedupon binding of PNA. These results indicate that in the TE buffer PNAoccupied all available binding sites on the DNA duplex efficientlydisplacing the DNA T-strand in the A98/T98 (SEQ ID NO:42) insert. WhenNaCl is added to the incubation buffer, the degree of conversion ofrcDNA into rapidly moving species dramatically decreased within a narrowrange between 20 and 40 mM of NaCl as seen in FIG. 17, panel A. Thisemphasizes a strong sensitivity of the PNA incorporation to the saltconcentration. However, after being formed at low salt, the complexshowed remarkable stability and tolerated increasing salt concentrationsup to at least 500 mm of NaCl (FIG. 20, panel B, lane 4). The complexcould be dissociated by alkali treatment (FIG. 20, panel B, lane 5).

EXAMPLE 18

Inhibition of RNA Polymerase T₃ Transcription Elongation by PNA

The complex, between PNA T₁₀ -LysNH₂ (SEQ ID NO:2) (1 or 10 uM) andpAIOKS (pBluescriptKS⁺ in which the d(A₁₀) target is cloned into theBamH1 site, analogous to pT10) (100 ng) cleaved with restriction enzymePvuII, XbaI, or BamH1, was formed by incubation in 14 μL 10 mM Tris-HCl,1 mM EDTA, (pH 7.4) buffer for 60 min. at 37° C. Subsequently 4 μL 5×concentrated polymerase buffer (0.2M Tris-HCl, pH 8.0, 125 mM NaCl, 40mM MgCl₂, 10 mM spermidine) was added together with 15 U T₃ RNApolymerase and ATP (10 mM), CTP (10 mM), GTP (10 mM), UTP (1 mM) and ³²P-UTP (0.1 μCi), and the incubation was continued for 10 min. Followingethanol precipitation, the RNA was analyzed by electrophoresis insequencing gels and the radiolabeled bands were visualized byautoradiography using intensifying screens. The results are shown inFIG. 18: Lane 1: pTIOKS was cut with PvuH and no PNA was present. Lane2: pTIOKS was cut with BamH1, and no PNA was present. Lane 3: pTIOKS wascut with XbaI, no PNA. Lane 4: as in lane 1 but in the presence of PNA(1 uM). Lane 5: as lane 3+PNA (1 uM). Lane 6: as lane 1+PNA (10 uM).Lane 7: as lane 3+PNA (10 uM).

As is seen in FIG. 18, In the absence of PNA the expected run-offtranscript is observed (lanes 1,2 & 3). When the preformed complexbetween PNA T₁₀ -LysNH₂ (SEQ ID NO:2) and the template was used, atranscript which corresponds in length to transcription arrest at theencounter of the PNA, i.e., one nucleotide longer than the run-off atthe BamH1 site (lane 3), is produced (lanes 4-7). This shows thattranscription elongation by RNA polymerase T₃ is effectively blocked byPNA bound to the template strand. If PNA was bound to the non-templatestrand, no transcript corresponding to blockage by the PNA was observed.Similar results were also obtained using RNA polymerase T₇ (data notshown). In this experiment the PNA/DNA complexes were formed in low saltbuffer prior to addition of the buffer required for enzyme action sincebinding of PNA to dsDNA is inhibited by elevated (>50 mM Na⁺) saltconcentrations. Once formed, the complexes are exceedingly stable tothese conditions.

EXAMPLE 19

Inhibition of Taq DNA Polymerase Primer Extension by PNA

A mixture of 100 500 ng Pvull cleaved plasmid pAIOKS, 0.1 ug M13 reverseprimer and 1 ug of PNA T₁₀ -LysNH₂ (SEQ ID NO:2) in 10 ul buffer (10 mMTris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.1 mg/ml gelatine) wasincubated for 5 min at 90° C. and then for 60 min at 37° C.Subsequently, 1 U of Taq polymerase, 1 ul dCTP (100 uM), dGTP (100 uM),dTTP (100 uM) and ³² P-DATP (1 uCi) were added and the incubationcontinued for 5 min at 20° C. Following the addition of 2 ul dATP, dCTP,dGTP, dTTP (1 mM each), the sample was incubated for 15 min at 60° C.The DNA was precipitated with ethanol and the samples treated asdescribed for Example 18. The results are shown in FIG. 19: Lane 1:pTIOKS was cut with PvuII and no PNA was present. Lane 2: same as laneone with PNA present. Lane 3: no PNA and pTIOKS was cut with BamH1.

As is shown in this example, in a primer extension experiment using apre-formed complex between PNA T₁₀ -LysNH₂ (SEQ ID NO:2), asingle-stranded target, and a primer down-stream from the targetsequence, a product corresponding in length to blockage at the PNA wasproduced by Taq DNA polymerase (FIG. 19, lane 2). No product wasdetected in the absence of PNA (lane 3). Similar results were obtainedusing the large fragment (Klenow fragment) of E. Coli DNA polymerase(data not shown). Thus demonstrates elongation by DNA polymerases isblocked by PNA.

EXAMPLE 20

Inhibition of Transcription by PNA

The PNA oligomers T₁₀ -LysNH₂ (SEQ ID NO:2), T₅ CT₄ -LysNH₂ (SEQ IDNO:6) and T₂ CT₂ CT₄ -LysNH₂ (SEQ ID NO:1) were synthesized as describedin Example 1. Plasmids containing the target sequences were obtained bycloning of the appropriate oligonucleotides into the vectorpBluescripKS⁺. To obtain pT10Ks and pA10Ks, 16-mers 5'-TCGACT₄ CT₅ G(SEQ ID NO:36) and 5'-GATCCA₁₀ G (SEQ ID NO:34) were cloned into theBamH1 site, and clones containing the insert in either orientation wereisolated. pT9CA9GKS was obtained by cloning 5'-TCGACT₄ CT₅ G (SEQ IDNO:36) and 5'-TCGACA₅ GA₄ G (SEQ ID NO:37) into the SalI site andpT8C2KS and pA8G2KS were obtained by cloning 5'-GT₄ CT₂ CT₂ CTGCA (SEQID NO:39) AND 5'-GA₂ GA₂ GA₄ CTGCA (SEQ ID NO:38) into the PstI site. E.coli JM103 was used as host in all cases, and transformations andisolation of clones were done by standard techniques. Plasmids werepurified by buoyant density centrifugation in CsCl gradients andcharacterized by dideoxy sequencing.

Inhibition of RNA Polymerase Transcription Elongation by PNA

The complex between the desired PNA and the desired DNA (100 ng) cleavedwith the desired restriction enzyme was formed by incubation in 14 μL 10mM Tris-HCl, 1 mM EDTA, (pH 7.4) buffer for 60 min at 37° C.Subsequently 4 μL 5× concentrated polymerase buffer (0.2M Tris-HCl, pH8.0, 125 mM NaCl, 40 mM MgCl₂ 10 mM spermidine) was added together with15 U of RNA polymerase and ATP (10 mM), CTP (10 mM), UTP (1 mM) and ⁼P-UTP (0.1 μCi). The incubation was continued for 10 min. Followingethanol precipitation, the RNA was analyzed by electrophoresis inpolyacrylamide sequencing gels, and radiolabeled RNA bands werevisualized by autoradiography (using Agfa Curix RPI X-ray films andintensifying screens). Quantitation was performed by densitometricscanning using a Molecular Dynamics laser scanner and the ImageQuant™software.

FIG. 20 illustrates certain concentration dependence between complexesof pA10Ks cleaved with XbaI and the following concentrations of PNA T₁₀-LsyNH₂ (SEQ ID NO:2): 0, 0.2, 0.35, 0.7, 1, 1.4, 1.75, 2.1, 2.8, 3.5,4.3 or 5 μM (lanes 1-12). The complexes were formed by incubation in TEbuffer for 60 min. at 37° C. The buffer was adjusted to transcriptionconditions and following the addition of NTPs, ³² P-UTP and T₃ RNApolymerase, transcription was allowed to proceed for 5 min. at 37° C.The transcripts were analyzed by electrophoresis in 10%polyacrylamide/7M urea gels and visualized by autoradiography.

The results shown in FIG. 21 are similar to those of FIG. 20 except thatthe plasmid pA8H2KS was cleaved with BamH1 and PNA T₂ CT₂ CT₄ -LysNH₂(SEQ ID NO:1) at the following concentrations: 0, 0.6, 1.2, 2.4, 3.6,4.8, 6, 7.2, 9.6, 14.4 or 16.7 μM (lanes 1-12).

FIG. 22 illustrates a quantitative representation of the results shownin FIGS. 20 and 21. In this figure the following symbols, respectively,are utilized, squares: pA10Ks×XbaI and PNA T₁₀ -LysNH₂ (SEQ ID NO:2);circles: pA8GKS×BamH1 and PNA T₂ CT₂ CT₄ -LysNH₂ (SEQ ID NO:1). closedsquares: relative amount of the truncated transcript; closed circles:total amount of transcript.

This examples illustrates that the phage T₃ and T₇ RNA polymerases canbe used as a model system for very efficient and robust transcription.In this example, a template was constructed by cloning the appropriatePNA targets into the polylinker of the BluescriptKS⁺ plasmid. The targetwas clone into the BamH1 site in both orientations, thus allowing forfour transcription experiments to be performed using either theconstructs and either the T₃ - or the T₇ -promoter. The resultspresented in FIGS. 20, 21 and 22 show the dose dependent inhibition ofthe transcription of pA10Ks (FIG. 20) or pA8G2KS (FIG. 21) by PNA T₁₀-LysNH₂ (SEQ ID NO:2) or T₂ CT₂ CT₄ -LysNH₂ (SEQ ID NO:1) using T₃ RNApolymerase. It is observed that as the concentration is raised theamount of full length transcript is decreased and the amount oftruncated product is increased suggesting that sequence specifictranscription arrest is taking place. However, the total amount oftranscript also decreases indicating that general inhibition oftranscription also occurs.

EXAMPLE 21

Sequence Specificity of the Transcription Elongation Arrest by PNA T₁₀-LysNH₂ and PNA T₂ CT₂ CT₄ -LysNH₂ using T₃ or T₇ RNA Polymerase

Samples identical to those used for Example 20 were analyzed byelectrophoresis in a polyacrylamide sequencing gel. The results areshown in FIG. 23 where, Lanes 1-4: pA10Ks/T₃ RNA polymerase; lanes 5-8:pT10 KS/T₇ RNA polymerase; lanes 9-12: pA8G2KS/T₃ RNA polymerase; lanes13-16: pT8C2KST7 RNA polymerase. The plasmid used in the samples oflanes 1-3 was cleaved with Xbal, that in lanes 6-8 with Pst1, that inlanes 9-11 with BamH1 and that in lanes 14-16 with HindIII. Lanes 1, 8,9 & 16 are controls without PNA. Lanes 4, 5, 12 & 13 are controlscleaved proximal to the PNA target site i.e., with BamH1 (lanes 4 & 5)or Pst1 (lanes 12 & 13). Samples of lanes 2, 3, 6 & 7 were preincubatedwith PNA T₁₀ -LysNH₂ (SEQ ID NO:2), and those of lanes 10, 11, 14 & 15were preincubated with PNA T₂ CT₂ CT₄ -LysNH₂ (SEQ ID NO:1).

In this example, in order to determine the position of transcriptionarrest, RNA transcripts were analyzed on high resolution sequencinggels. The results show that a specific RNA transcript is produced by T₃-RNA polymerase in the presence of PNA-T₁₀ (SEQ ID NO:2) that is onenucleotide longer than the run-off transcript produced using a template(BamH1 cleaved) ending one nucleotide in front of the PNA target (FIG.23, lanes 2-4). Thus transcription proceeds to, but does not include,the first nucleotide involved in hydrogen bonding with the PNA. Theresults obtained with T₇ -RNA polymerase are similar to those with T₃-RNA polymerase (FIG. 23, lanes 5-8). Interestingly, however, in thiscase the transcript is less homogeneous in length than the transcriptobtained with the T₃ -polymerase, and the lengths of the transcriptsindicate that the polymerase is able to transcribe into the PNA target.These results show that this polymerase is able to some extent todisplace the bound PNA during transcription, although the polymerase-PNAencounter may sometimes result in chain termination. Using mixed A/G PNAtargets gave the results shown in FIG. 23, lanes 9-16. It is noteworthythat transcription arrest with an A₄ GA₅ (SEQ ID NO:43) target and a T₄CT₅ -LysNH₂ (SEQ ID NO:8) PNA occurs one to two nucleotides inside thetarget, while transcription arrest with an A₄ GA₂ GA₂ (SEQ ID NO:44)target and a T₄ CT₂ CT₂ -LysNH₂ (SEQ ID NO:9) PNA is much less efficient(as compared to the results of Example 22) and occurs at the end of thetarget (FIG. 23, lanes 10, 11, 14 & 15).

EXAMPLE 22

Transcription Arrest in pT9CA9GKS Plasmid

In FIG. 24 the effect of PNA T₁₀ -LysNH₂ (SEQ ID NO:2) (lanes 1, 2, 4 &5) on T₇ RNA polymerase transcription of pT10 KS (lanes 1-3) and pA10Ks(lanes 4 & 5), and of PNA T₅ CT₄ -LysNH₂ (SEQ ID NO:6) (lanes 6, 7, 10 &11) on T₃ or T₇ RNA polymerase transcription of pT9CA9GKS (lanes 6-11)are shown. The plasmid of the samples in lanes 1, 2, 4-7, 10 & 11 werecut with PvuII while BamH1 was used for the sample of lane 3, and SalIwas used for the samples of lanes 8 & 9. The PNA concentration was 5 μM.

As is shown in this example, experiments using the pT9CA9GKS plasmid andT₃ or T₇ RNA polymerase showed (FIG. 26, lanes 5-11) that transcriptionarrest at this PNA target occurred 2-3 bases inside the target.Employing a gel-retardation assay, it was ascertained that the complexesbetween PNAs T₁₀ -LysNH₂ (SEQ ID NO:2), T₄ CT₅ -LysNH₂ (SEQ ID NO:8) orT₄ CT₂ CT₂ -LysNH₂ (SEQ ID NO:9) with their complimentary dsDNA targetsare stable for at least 15 min when transferred from the TE-buffer tothe transcription assay-buffer, thus eliminating the possibility thatthe PNA was dissociating spontaneously during transcription. Theseresults indicate that an increase in the G-content of the target, andthus in the C-content of the PNA decreases the efficiency oftranscriptional arrest due to read-through. In all cases it is alsoobserved that PNAs at higher concentrations result in an unspecificinhibition of transcription by T₃ or T₇ RNA polymerase. We cannot say ifthis effect is due to an inhibitory effect of the PNA directly at thepolymerase, or if it is caused by binding of the PNA to other sites onthe DNA template, e.g., the promoter.

EXAMPLE 23

Sequence Specificity of the Transcription Elongation Arrest by PNA T₁₀-LYsNH₂

In this example, template versus non-template binding of the PNA wasexamined. The experiments were performed essentially as described forExample 21. The results are shown in FIG. 25 using plasmid pA10Ks (a &c) or pT10KS (b) cleaved with PvuII (lanes 1 & 4), Xbal (lanes 2 & 5) orBamH1 (lane 3). PNA T₁₀ -LysNH₂ (SEQ ID NO:2) was present at 3.3 μM inthe samples of lanes 4 & 5. Panel c is a longer exposure of panel a.

As is shown in FIG. 25, if the PNA is bound to the non-template strandtranscription is virtually not arrested (FIG. 25, FIG. 24: lanes 1-5). Avery weak band corresponding to arrest at the end of the PNA target canbe detected upon longer exposure of the autoradiogram (FIG. 25, panel c,lanes 4,5). These findings are similar to results reported with DNAtemplates site specifically modified with covalent psoralen adducts.

EXAMPLE 24

Sequence Specificity of the Transcription Elongation Arrest by PNA T₅,T₇ or T₁₀ -LysNH₂ using pA10Ks/T₃ RNA Polymerase

In this example transcription arrest with PNAs shorter than 10 mers wasexamined. For the results shown in FIG. 26, the plasmid was cleaved withXbaI in all cases and preincubated with PNA T₆ -LysNH₂ (SEQ ID NO:7)(μM, lane 2), T₇ -LysNH₂ (SEQ ID NO:45) (μM, lane 3) or T₁₀ -LysNH₂ (SEQID NO:2) (μM, lane 4). Lane 1 is a control without PNA.

The results seen in FIG. 26 show that an 8-mer and even a 6-mer PNA,although less efficiently, are able to arrest transcription by T₃ -RNApolymerase. The results also show that the arrest is occurring with thePNA bound at the far end of the target indicating that the PNA isbinding to the 10-mer target in a floating mode and that the RNApolymerase is "pushing" the PNA.

EXAMPLE 25

Transcription Initiation from PNA-DNA Displacement Loops

Peptide nucleic acid (PNA) form (PNA) 2/DNA, DNA triplex-D-loopstructure upon binding to complimentary double-stranded DNA as shownschematically in FIG. 27. If two adjacent PNA sites are present in cisor in trans (wherein cis is where two PNA bind adjacent to each other onthe same strand and trans is where the two PNA bind on oppositestrands), structures of the type shown in FIG. 27 b,c are formed. Inthis example, since these structures resemble transcription elongationloops we tested if E. coli RNA polymerase is able to recognize and bindto such PNA/DNA loops.

The three plasmids pT9C, pT9CT9C. (pUC19 derivatives) and pT9CA9GKS(Bluescript derivative) were used for this in vitro transcriptionexperiments. Restriction fragments were isolated by digestion with PvuIIand on polyacrylamide gels resulting in the fragments shown in FIG. 28.In addition, the isolated PvuII fragment from pKST9C was restricted withXbaI or SacI before PNA hybridization to obtain a shorter transcriptfrom the `PNA-promoter`. PNA-DNA complexes were formed by combining 0.3,μ PNA (50 OD) with DNA fragments in 10 mM Tris-HCl pH 8.0 and 0.1 mMEDTA in a total volume of 25 μL for 1 hour at 37° C. The transcriptionwere initiated by addition of 50-100 mM E. coli RNA Polymeraseholoenzyme, T3 and T7. The reaction mix contained a final concentrationof: 40 mM Tris-HCl pH 8.0, 120 mM KCl, 5 mM MgCl₂, 0.1 mM DTT and 1 mMof ATP, CTP, GTP and 0,1, mM of UTP and ³² p UTP. The complete mixtureof 30 ul were incubated at 37° for 20 minutes followed by ethanolprecipitation. The RNA transcripts were analyzed on 8% denaturingpolyacrylamide gels, and visualized by autoradiography. The RNAse Hexperiment was done by hybridization of a complimentary oligonucleotideto the mRNA to synthesize a double-stranded target for RNAse H.

EXAMPLE 26

Cis Competition between the PNA Promoter and lacUV5

A PvuII fragment including a single or a triple PNA binding sitetogether with the lacUV5 promoter was isolated. The DNA was incubatedwith increasing amounts of PNA for 1 hour. The transcriptions wereperformed as described in Example 25. As indicated by the gel-shiftsshown in FIG. 29, a slower migrating complex between the target DNA andthe RNA polymerase is formed only if a PNA binding to this target isformed is prebound to the DNA.

EXAMPLE 27

RNA Polymerase Footprinting

In this example DNase I footprinting experiments are undertaken todefine where on the DNA fragment binding takes place. DNA fragments ofthe length shown in FIG. 30 are labeled in the 3' end with klenowpolymerase and ³² P-dXTP. The PNA-DNA complexes are formed as describedin Example 26 above. The RNA Polymerase-PNA/DNA complexes are formed ina reaction buffer as described for Example 26 with the addition of 2ug/ml calf Thymus DNA in a total volume of 100 ul. After 15 min.incubation at 37° C. the samples are digested with 0.03 μL DNAse (1mg/ml) for 3 min followed by ethanol precipitation and analyzing by 8%denaturing PAGE. Expected results of this example are also shown in FIG.29. In the presence of PNA but in the absence of RNA polymerase only aweak footprint corresponding to PNA binding to the target is observed.However, upon addition of E. coli RNA polymerase a clear footprint isobserved.

EXAMPLE 28

Gel-Shift Assay

PNA-DNA complexes are formed as for the footprint experiment in Example27. After 15 min at 37° C. incubation the samples are analyzed on 5%PAGE. The gel-shift and DNaseI footprinting results should demonstratethat E. coli RNA polymerase binds to a PNA/dsDNA strand displacementloop. The binding should differ distinctly from that seen for RNApolymerase in the initiation and in the elongation complex. Binding incase of the PNA/DNA complex likely will be confined to the DNA loop andwill not extend much into the surrounding double-stranded DNA.

Although the (PNA)₂ /DNA,DNA triplex-D-loop structurally does resemblean RNA transcription elongation loop, it differs in one importantaspect; the PNA does not contain a 3'-hydroxyl group to be used as anelongation substrate for RNA polymerase. Nevertheless, PNA dependenttranscription is observed from a DNA molecule containing a PNA target.Furthermore, the length of the resulting transcript should correspondsto a run-off transcript initiated at the bound PNA, as exemplified inFIG. 27a. The transcription should be more efficient if a double PNAtarget is used giving rise to a loop of approximately 30 bases (FIG.27b) in the cis configuration, and approximately 16 bases in the transconfiguration (FIG. 27c). In the latter case, transcripts of twodistinct sizes should be produced (FIG. 27c), which in lengthcorresponds to initiation at both PNA targets and proceeding in oppositedirections (FIG. 27c).

Two experiments will be undertaken to estimate the strength of the PNAdependent transcription initiation. In one experiment, both the PNAtarget and the strong CAP independent UV5 promoter are present on thesame DNA fragment. Upon titration with PNA the full run-off transcriptfrom the UV5 promoter is inhibited, while a new transcript correspondingto transcription arrest at the PNA site appears. A very faint band of alength corresponding to transcription initiated at the PNA site is alsoobserved. However, when a DNA fragment containing a triple PNA bindingsite is used, transcription from this "PNA-promoter" should be able tocompete fully with the UV5 promoter. As the PNA concentration isincreased, transcription from the UV5 promoter decreases withconcomitant increase in the amounts of two transcripts that correspondin length to the products expected for transcription in either from the"PNA-promoter". An analogous experiment should be performed in transusing a mixture of a UV5 containing DNA fragment and either of the DNAfragments containing the single or the cis or trans double PNA targets.The results of these experiments should confirm that a single PNAdecamer target is not able to compete with the UV5 promoter whereas bothof the dimeric targets competes very efficiently. While we do not wantto be bound by theory, the results should suggest that a single-strandedDNA loop is a major structural determinant for RNA polymerase upontranscription initiation and elongation.

EXAMPLE 29

Site Specific Termination of in Vitro Transcription in EukaryoticNuclear Extracts

PNA TTTTCTTTT-NH₂ (SEQ ID NO:16), TTCCCTTCC-LysNH₂ (SEQ ID NO:12),CCCCCCCTTTTTTTTT-NH₂ (SEQ ID NO:18), TTCTCTCTCT-NH₂ (SEQ ID NO:20) andCCTCCTTCCC-NH₂ (SEQ ID NO:19) were synthesized as described inExample 1. Phosphodiester oligonucleotides were synthesized usingstandard protocols as for example Vickers, T. et. al., Nucleic AcidsResearch, 1991, 19, 3359-3368. Plasmids were constructed with the CMV IE1 promoter driving the transcription of viral DNA sequences. The plasmidpIE72 is a CMV IE cDNA clone which was constructed as described byStenberg, M. et. al., J. Virol., 1993, 64, 1556-1565. pCH495 wasconstructed by ligating the 495 bp SalI/EcoRI fragment from the plasmidpBH10 (see Hahn, B., et. al., Nature, 1984, 312, 166-169) into thevector pUC-CMV, prepared by digestion with the same two enzymes. pCINwas constructed by ligating a 463 bp PvuII fragment from the plasmidpSVCC3 (see Depeto, A. S. and Stenberg, R. M., J. Virol., 1989, 63,1232-1238), a genomic CMV clone, into the vector pCEP-4 (Invitrogen)linearized at the PvuII site. pCINr was constructed in the same manner,however the insert is cloned in the opposite orientation. The plasmidsare illustrated in FIG. 31. All plasmids were constructed with the CMVIE1 promoter (open box) driving the transcription of sequencescontaining homopurine sites for targeting with homopyrimidine PNA.pCH495 contains a 495 bp SalI/EcoRI fragment from pBH10 (3738 to 4233 ofthe HIV-1 genome). The plasmid pCIN contains a 463 bp PvuII fragmentsubcloned from the plasmid pSVCC3, a genomic CMV clone. pCINr containsthe same fragment in the opposite orientation. pIE72 is a CMV IE1 cDNAclone. The filled boxes represent the transcribed region of eachlinearized plasmid. The expected transcript length is shown at the 3'end of each. The sequence of the PNA target site is also shown alongwith the position relative to the end of the transcript. In vitrotranscription. 100 ng/ul of linearized plasmid was pre-incubated withPNA at various concentrations in a volume of 10 ul for 3 hours at roomtemperature in KCl buffered with 10 mM HEPES, pH 6.8. For studies on pHdependence, KCl was buffered with 10 mM TrisOAc, pH 5.1 or 10 mMTris-HCl, pH 7.9. KCl ranged in concentration from 0.5 to 100 mM.Following the pre-incubation, 2 ul of the PNA bound plasmid wastranscribed in HeLa nuclear extracts (Promega) following themanufacturer's protocol. The transcribed RNA's were precipitated thenseparated by electrophoresis through 5% denaturing acrylamide gels, run2 hours at 15 W. Gels were then dried and exposed to film. Bands on theexposed film were quantitated using a Molecular Dynamics laserdensitometer.

A PNA with the sequence TTTTCTTTT-NH₂ (SEQ ID NO:16) and a nonhomologous PNA control were pre-incubated with the 200 ng of EcoRIlinerized plasmid pCH495 at the indicated concentrations for 3 hours at22° C. in 10 ul of 10 mM KCl, pH 6.8 (potassium salt was used in allbinding buffers because it is the most compatable with the transcriptionextract). Following the pre-incubation, the PNA bound plasmid wastranscribed in HeLa cell nuclear lystates (Promega) containing ³² p GTP.The transcribed RNA product was then electrophoresed on a 5% acrylamidegel with 50% w/v urea, which was then dried and exposed to film. Theexpected size of the full length run off transcript and the PNAtruncated transcript are shown in FIG. 31. The concentration at which50% of the transcript was truncated was determined to be approximately80 uM by quantitating the bands from the exposed film using a MolecularDynamics densitometer. Results are shown in FIG. 32. Linearization ofthe plasmid with EcoRI and PvuII provides transcription templates withexpected run-off transcripts of 495 (lane 1) and 190 (lane 2)nucleotides respectively. In the presence of the complementary PNA, theEcoRI linearized plasmid yields two distinct transcripts, onecorresponding to the run-off transcript and a second truncatedtranscript with a molecular weight consistent with the 373 nucleotideproduct expected if truncation occurred at the PNA binding site. Therelative proportion of the truncated nucleotide product increases withincreasing concentration of PNA in the pre-incubation. When a DNAoligonucleotide of the same sequence (5'-TTTTCTTTT-3') was used in placeof the PNA no truncated product was observed (data not shown). Theexperiment was repeated for the other PNA target sites shown in FIG. 31.These results are shown in Table I and are given as IVT₅₀ ; the PNAconcentration at which 50% of the transcript is specifically truncated.All pre-incubations were carried in 10 mM KCl at pH 5.1, 6.8, or 7.9. Inall cases, the amount of transcript, both full length and truncated,decreases with increasing PNA concentration, suggesting a non-specificinhibition of transcription by PNA. This effect seems to be become morepronounced as the percentage of cytosine residues in the PNA increases.For example, at a PNA concentration of 1 mM, the total yield oftranscript is reduced approximately 63 percent in the presence of PNATTTTCTTTT-NH₂ (SEQ ID NO:16), 84 percent in the presence ofTTCTCTCTCT-NH₂ (SEQ ID NO:20) and completely in the presence ofCCCCCCCTTTTTTTTT-NH₂ (SEQ ID NO:18) (compare the 1 mM lanes in FIG. 32and FIG. 33. Direct addition of PNA to transcription extracts did notresult in the production of any truncated product (data not shown).

                  TABLE I                                                         ______________________________________                                        Summary of in vitro transcription activity for PNA oligomers.                 PNA            plasmid  pH 5.1  pH 6.8                                                                              pH 7.9                                  ______________________________________                                        TTTTCTTTT-NH.sub.2                                                                           pCH495   58 uM    76 uM                                                                              >500 uM                                 TTCTCTCTCT-NH.sub.2                                                                          pIE72    29 uM   103 uM                                                                              >1 mM                                   CCCCCCCTTTTTTTTT-NH.sub.2                                                                    pCIN,    30 uM   124 uM                                                                              >1 mM                                                  pCINr                                                          CCTCCTTCCC-NH.sub.2                                                                          pIE72    >1 mM   none  none                                    ______________________________________                                    

EXAMPLE 30

Effect of Target Site Orientation on PNA Mediated Termination of inVitro Transcription.

Plasmids PCIN and pCINr were linearized with HindIII, then pre-incubatedwith PNA of the sequence CCCCCCCTTTTTTTTT-NH₂ (SEQ ID NO:18) at theconcentrations shown in FIG. 33 under the conditions described forExample 29. Following the IVT a truncated product was clearly observedfor pCIN, where the PNA is bound to the template strand. However, forpCINr, where the PNA is bound to the non-transcribed strand, only aslight amount of truncated product of the expected size is observed. Theplasmids pCIN and PCINr contain a CMV gene fragment cloned in oppositeorientations under the transcriptional control of of the CMV promoter.PCIN contains the binding site for the PNA CCCCCCCTTTTTTTTT-NH₂ (SEQ IDNO:18) on the template strand, while pCINr contains the same target siteon the non-transcribed strand. The PNA was tested for the ability toeffect in vitro transcription as per Example 29. The results are shownin FIG. 33. Both plasmids show non-specific inhibition of in vitrotranscription at the highest concentration of PNA tested (1 mM).However, when the PNA concentration is decreased to 100 uM,approximately one half of the RNA produced corresponds to the sizeexpected for the PNA truncated product (209 nucleotides) with the pCINtemplate. In contrast, pCINr shows only a faint truncated product of thecorrect size at the same concentration.

EXAMPLE 31

Effect of Ionic Strength and pH on PNA Mediated Termination ofTranscription.

25 uM PNA TTTTCTTTT-NH₂ (SEQ ID NO:16) was pre-incubated three hourswith pCH495 at pH 6.8 in 0.5 to 100 mM KCl buffer. Following thepre-incubation the linearized plasmid was transcribed as detailed inExample 29. The results are shown in FIG. 34 with the percent PNAtruncated product indicated at the bottom of each lane. The proportionof the truncated product relative to full length transcript is increasedas the salt concentration is decreased.

The same PNA and plasmid template were used to determine the effects ofpH on the ability of PNA to effect transcription. PNA at a concentrationof 10 or 100 uM, was pre-incubated (as described in Example 29) with thetemplate in 10 mM KCl buffer at pH 5.1, 6.8, or 7.9 then transcribed inHeLa cell extracts. The resultant transcription products are shown inFIG. 35 with the percent truncated product shown below each lane. At thehighest pH tested, 7.9, little specifically truncated product isobserved even at a PNA concentration of 100 uM. A slight decrease in pHto 6.8 results in a dramatic increase in the amount of truncated messageproduced at the 100 uM PNA concentration, although no truncated messageis observed at a PNA concentration of 10 uM. However, when the pH isdecreased to 5.1, a small amount of the truncated product is observedeven at the lower PNA concentration and is increased at the higher PNAconcentration.

The experiments were also performed with the other PNA's. The IVT₅₀ foreach is listed in Table I, above. The effect of pH on PNA binding seemsto become more enhanced as the percentage of cytosine residues in thePNA oligomer increases, probably due to enhanced binding upon cytosineprotonation. At pH 5.1, one PNA, CCTCCTTCCC-NH₂ (SEQ ID NO:19), showedonly a small amount of truncated product at the highest concentrationtested. At higher pH no truncated product was observed at all. HigherPNA concentrations were not tested since they resulted in completenon-specific inhibition of transcription. This non-specific effect wasgenerally more pronounced in the oligomers with largercytosine/thymidine ratios.

The ability of PNA to inhibit transcription in vitro supports it use asan antigene therapeutic agent. In using PNA as an antigene agent both pHand ionic barriers would be considered. The effects of pH are mitigatedby targeting adenosine rich regions, avoiding dG/dC duplexes.Alternatively, modified residues, such as methyl cytosine couldfacilitate binding. In this example inhibition of in vitro transcriptionwas most efficient at ionic strengths less than those typically found incells. Therefore, it is possible that PNA will strand invade underphysiological conditions if given sufficient time to overcome kineticbarriers. Further to enhance the rate of strand invasion metabolicallyactive regions of the genome would be targeted.

EXAMPLE 32

In Vitro Binding of PNA to Target.

The target for in vitro binding of duplex DNA was prepared by annealingtwo 50 base DNA oligonucleotides in which the binding site for PNATTTTCTTTT-NH₂ (SEQ ID NO:16) is centered (5'-AAACAGGGCA GGAAACAGCATATTTTCTTT TAAAATTAGC AGGAAGATGG-3' and 5 '-CCATCTTCC TGCTAATTTTAAAAGAAAAT ATGCTGTTTC CTGCCCTGTTT-3' (SEQ ID NO:46)). The target was ³²P end labeled, then incubated at roughly 500 nM with variousconcentrations of PNA in the buffers described above for 4 hours at roomtemperature. PNA bound duplex was seperated from free by electrophoresison a 5% native acrylamide gel in TBE. The gel was then dried and exposedon a Molecular Dynamics PhosphoImager. The K_(d) is given as the PNAconcentration at which one half of the labeled target is bound. Gelshifts of PNA against DNA oligonucleotide complement were carried out inthe same buffers using an end labeled DNA oligonucleotide with thesequence 5-AAAAGAAAA-3', which was present in each hybridization mix at1 nM. Bound DNA oligonucleotide was seperated from free byelectrophoresis through a 12% native acrylamide gel in TBE.

Following the above protocols, the in vitro binding of PNA to DNA wasassessed by gel mobility shift assay. The 50 base pair duplex DNA targetfor the PNA TTTTCTTTT-NH₂ (SEQ ID NO:16) was prepared with the bindingsite for PNA TTTTCTTTT-NH₂ (SEQ ID NO:16) centered in the fragment. TheDNA complement (5'-AAAAGAAAA-3') was synthesized and end labeled. PNAwas incubated with the either the single strand complement or duplex DNAtarget in 20 mM KCl buffer at pH 5.1, 6.8, or 7.9. PNA bound DNA wasthen seperated from free on a native acrylamide gel. FIG. 36a shows thatPNA binding to both the single stranded and duplex target is effected bythe pH of the binding buffer. Binding affinity (K_(d), the PNAconcentration at which one half of the target is shifted in mobility) toboth single stranded and duplex DNA complement was determined by the gelshift assay. As the pH is decreased affinity increases. PNA affinity forthe single stranded target is much greater than for the duplex target,presumably due to the stability of the DNA duplex in 20 mM KCl. Otherexperiments to determine the binding affinity of other PNA oligomersshowed that the effect of pH on binding was more pronounced withcytosine rich PNA's (data not shown).

Binding of PNA to target was also utilized to study the effects of saltconcentration on PNA binding by incubating PNA TTTTCTTTT-NH₂ (SEQ IDNO:16) with it's single stranded or duplex target in buffers containing1, 10, or 100 mM KCl at pH 6.8. The results are shown in FIG. 36b. Whilethe salt concentration does have a large effect on the ability of PNA tobind duplex DNA, there is little salt effect on the binding to singlestranded complement. Similar results were obtained when PNATTCCCTTCC-LysNH₂ (SEQ ID NO:12) was tested (data not shown).

EXAMPLE 33

PNA-DNA Base Pair Recognition

To test if PNA containing all four natural nucleobases is a true DNAmimic in terms of base pair specific hybridization to complementaryoligonucleotides a pentadecamer PNA was designed to contain an almostequal number of pyrimidines and purines yet having no more than twopurines or pyrimidines juxtaposed. It contains an equal number ofthymines and cytosines and a single guanine. Furthermore, the sequenceof the pentadecamer is non-selfcomplementary and contains a GTCAsequence at the center. By measuring the thermal stability of complexesbetween the PNA pentadecamer and the Watson-Crick complementaryoligonucleotide, as well as 12 other oligonucleotides each having asingle base mismatch at one of the four center PNA nucleobases,information about PNA-DNA base pair recognition was obtained. Finally,the PNA was designed with no pyrimidine stretches so as to stronglydisfavor--if not prohibit--triplex formation.

The thermal stability measured as the melting temperature, Tm, ofcomplexes between the pentadecamer, H-TGTACGTCACAACTA-NH₂ (SEQ ID NO:10)and the complementary deoxyoligonucleotide 3'-ACATGCAGTGTTGAT (SEQ IDNO:47) (termed anti-parallel orientation: amino terminal of the PNAcomplementary to the 3'-end of the oligonucleotide) was 69.5° C. (seeTable II), whereas the T_(m) of the corresponding PNA-DNA complex inparallel orientation was 56.1° C. The T_(m) for the PNA-RNA complexeswas 72.3 and 51.2° C., respectively (see Table II). The orientationpreference was further settled by using two decamer PNAs, andhybridizing these to complementary oligonucleotides in bothorientations. The anti-parallel orientation was preferred in all cases(see Table II). Furthermore, it is noteworthy that virtually identicalT_(m) s were obtained regardless of which strand is the PNA and which isthe DNA (see Table II, 2nd & 3rd row). The results also show that thepresence of a terminal lysine amide (add to certain PNAs to reduceaggregation of oligo thymine PNA) does not influence the preferredorientation of the PNA relative to the DNA.

                                      TABLE II                                    __________________________________________________________________________    Melting temperatures T.sub.m (°C.) for PNA/DNA, PNA/RNA, DNA/DNA       and DNA/RNA complexes..sup.a                                                  1st strand sequence.sup.b                                                              TGTACGTCACAACTA.sup.c                                                                     GTAGATCACT.sup.d                                                                       AGTCATCTAC.sup.d                                __________________________________________________________________________    DNA:DNA  53.3        33.5     33.5                                            DNA:RNA  50.6        nd       nd                                              PNA:DNA  56.1        38.0     38.0                                            (parallel)                                                                    PNA:DNA  69.5        51.0     49.0                                            (anti-parallel)                                                               PNA:RNA  51.2        nd       nd                                              (parallel)                                                                    PNA:RNA  72.3        nd       nd                                              (anti-parallel)                                                               __________________________________________________________________________     .sup.a Absorbance vs. temperature curves were measured at 260 nm in 100 m     NaCl, 10 mM Naphosphate, 0.1 mM EDTA, pH 7. T.sub.M, the temperature at       which half of the molecules are hybridized was obtained by fitting            triplicate melting curved at 4 μM of each strand to a modified two         state model with linear sloping baseline.                                     .sup.b Written 53' for oligonucleotides and N-- to C-- terminal for PNA.      .sup.c The PNA terminates in a carboxamide.                                   .sup.d The PNA terminates in a lysine amide.                             

When a Watson-Crick base pair mismatch was introduced in theoligonucleotide at any position facing the four middle PNA nucleobases(GTCA) in the pentadecamer a large increase in T_(m) (8-20° C., FIG. 37,was observed, thereby providing compelling evidence that PNA-DNArecognition takes place by Watson-Crick base pairing, i.e., A-T and G-Cbase pairing. Qualitatively similar results were obtained usingoligonucleotides in the parallel orientation. Shown in FIG. 37 are theeffect of base pair mismatches on the thermal stability of PNA/DNAcomplexes. Thermal stability of complexes between PNA,H-TGTACGTCACAACTA-NH₂ (SEQ ID NO:10) and the thirteen oligonucleotides3'-d(ACATGXYZVGTTGAT), in which X,Y,Z,V=C,A,G,T for the case where thePNA and DNA sequences are complementary are shown. In each of the twelveother oligonucleotides, three of the bases (X,Y,Z,V) were complementarywhile the fourth was one of the three non-complementary nucleobases. Forexample, when (X=T,Y=G,Z=A) then V=A or C. or G etc. Thus each of thetwelve oligonucleotides contains one of the twelve possible base pairmismatches relative to the PNA pentadecamer. The T_(m) of thesecomplexes are displayed as solid bars in FIG. 37. For comparison, theresults of similar experiments performed with DNA/DNA duplexes aredisplayed (hatched bars). Hybridizations were performed in 10 mMNa-phosphate, pH 7.0, 150 mM NaCl, 1 mM MgCl₂.

For comparison we also measured the thermal stabilities of thecorresponding DNA/DNA duplexes as shown in FIG. 38. ΔT_(m) values formismatches are shown in FIG. 38. Hybridizations were as for FIG. 37.

It is noteworthy that for virtually all base pair mismatched, thedecrease in stability is greater for the PNA/DNA complex than for theDNA/DNA complex (see FIG. 38), thereby indicating that the sequencediscrimination is, if anything, more efficient for PNA recognizing DNAthan for DNA recognizing DNA.

The unambiguous evidence for Watson-Crick base pairing suggest thatthese PNA/DNA complexes are duplexes rather than the (PNA)₂ /DNAtriplexes previously observed with homo-pyrimidine PNA. This conclusionwas confirmed by titration experiments using ³² P-labeledoligonucleotides in a gel retardation assay. Complete complex formationwas observed at a 1:1 stoichiometry of [PNA] to [DNA] as shown in FIG.39. Similar results were obtained using circular dichroism for thedetection of complex formation (data not shown). In this exampletitration was by gel-shift of the binding of PNA H-TGTACGTCACAACTA-NH₂(SEQ ID NO:10) to the 5'-endlabeled oligonucleotide3'-d(ACATGCAGTGTTGAT) (SEQ ID NO:47). The oligonucleotide was labeledwith ³² P at the 5'-end using standard techniques²². The oligonucleotide(1 nmole, 10³ cpm) was incubated with various amounts of PNA (0-5 nmol)in 10μ 10 mM Tris-HCl, 1 mM EDTA, pH 7.4 for 1 hour at 37° C. Thesamples were analyzed by electrophoresis in 20% polyacrylamide gels (TBEbuffer=89nM Tris-borate, pH 8.3, 1 mM EDTA) and the radiolabeled DNAvisualized by autoradiography. Concentrations of oligonucleotides andPNA were measured photometrically. Similar results were obtained usingthe complementary oligonucleotide of reversed polarity(5'-d(ACATGCAGTGTTGAT) (SEQ ID NO:52).

EXAMPLE 34

Structure of the PNA/DNA Duplex

The T_(m) data (Table II above) show that PNA, in contrast to DNA orRNA, may bind to complementary DNA or RNA in either orientation,although the antiparallel orientation is preferred. Since the PNAbackbone is achiral, the orientation does not by itself impose anysteric constraints on the winding of the helix, i.e., left or righthanded helicity, and computer modelling also indicates that both bindingorientations are possible.

Information on secondary structure may be obtained from circulardichroism (CD) measurements since these are sensitive to the base pairgeometry in the helix. FIG. 40 shows the CD spectra of the PNA/DNA,PNA/RNA, DNA/DNA and DNA/RNA duplexes. Shown in FIG. 40 is the circulardichroism spectra of PNA/DNA (a: antiparallel; b: parallel), PNA/RNA (c:antiparallel; d:

parallel), DNA/DNA (e) and DNA/RNA (f) complexes. The complexes wereformed by mixing equal molar amounts of the two complements in distilledH₂ O. Circular dichroism spectra were recorded on a Jasco 700 instrumentat room temperature using an optical path of 1 mm. All measurements wereaveraged ten times and smoothed. All spectra are largely similarsuggesting that PNA/DNA and PNA/RNA duplexes are right handed heliceswith a base pair geometry not drastically different from that found in aB- or an A-DNA helix. However, it is interesting that the CD-spectra,and thus the structure, of PNA/DNA (or PNA/RNA) duplexes are distinctlydifferent when parallel and anti-parallel complexes are compared. Thereference DNA/DNA duplex would be expected to adopt a B-like helix whilethe RNA/DNA duplex would be expected to adopt a more A-like helix, butthe CD-spectra of neither of these short duplexes show typical B-orA-like features. Thus it is not possible to conclude from these resultsif the PNA/DNA or PNA/RNA helices are preferentially A- or B-like.

EXAMPLE 35

Thermodynamics of the PNA/DNA Duplex Formation

Thermodynamics parameters for hybridization can be extracted fromthermal stability measurements when these are performed at varyingconcentrations of the complexes, or from the shape of the thermaldenaturation curves. Both methods were used to determine ΔH°, ΔS° andΔG° for formation of the PNA/RNA, DNA/RNA, PNA/DNA and DNA/DNA duplexes(Table III). It is remarkable that the decrease in entropy is almostidentical for the formation of DNA/DNA and PNA/DNA duplexes and thatboth reactions are strongly enthalpy driven.

                  TABLE III                                                       ______________________________________                                        Thermodynamic parameters for the formation of PNA/DNA,                        PNA/RNA, DNA/RNA and DNA/DNA duplexes with the sequence                       TGTACGTCACAACTA present in the PNA strand strand.sup.a.                               DNA:RNA PNA:RNA  DNA:DNA   PNA:DNA                                    ______________________________________                                        ΔH° (kcal/mol).sup.b                                                       -94.0     -109.7   -91.5   -103.5                                   ΔS° (eu).sup.b                                                             -264.9    -291.4   -253.9  -276.4                                   ΔG° .sub.3.sup.b                                                           -11.8     -19.3    -12.7   -17.7                                    T.sub.M (°C., 8 μM).sup.b                                                     49.8      72.3     53.6    68.9                                     ΔH° (kcal/mol).sup.c                                                       -128.9    -128.5   -105.3  -106.6                                   ΔS° (eu).sup.c                                                             -372.8    -345.9   -296.2  -285.8                                   ΔG°.sub.37 (kcal/mol).sup.c                                                -13.3     -21.2    -13.4   -18.0                                    T.sub.M (°C., 8 μM).sup.c                                                     50.1      72.2     53.5    68.8                                     ΔH° (kcal/mol).sup.d                                                       -111.5    -119.1   -98.4   -105.0                                   ΔS° (eu).sup.d                                                             -318.9    -318.7   -275.1  -281.1                                   ΔG°.sub.37 (kcal/mol).sup.d                                                -12.6     -20.2    -13.1   -17.9                                    T.sub.M (°C., 8 μM).sup.d                                                     50.0      72.2     53.5    68.8                                     ______________________________________                                         .sup.a Measured in 100 mM NaCl, 10 mM Na-phosphate, 0.1. mM EDTA, pH 7.0.     .sup.b Obtained by fitting melting curves to a modified two state model       with linear sloping baselines.                                                .sup.c Obtained from linear plots of 1/T.sub.M versus log (concentration)     .sup.d Temperature independent parameters calculated as the average of th     two methods described above.                                             

The finding that ΔH° and ΔS° are similar for the formation of PNA/DNAand DNA/DNA duplexes, combined with the finding that the PNA/DNA andRNA/DNA duplex structures have similar stacking according to the CDresults, indicates that single stranded PNA must have much the samedegree of base stacking as single stranded DNA, and thus appears to behighly structured. The kinetics of PNA/RNA duplex formation was alsomeasured, and the results show that the rate of hybridization is atleast as fast as that for 2'O-Me-RNA/RNA or DNA/DNA duplex formation(Table IV). Again fully consistent with the suggestion that the singlestranded PNA is at least as prestructured for duplex formation as is DNA(or RNA).

                  TABLE IV                                                        ______________________________________                                        Equilibrium and rate constants for duplexes.                                  Duplex    [Na+]     (M)T(°C.)                                                                       K.sub.D.sup.a                                                                         k.sub.1 (M.sup.-1 s.sup.-1).sup.a,b      ______________________________________                                        PNA/RNA.sup.c                                                                           0.1       37       5 × 10.sup.-11                                                                  2 × 10.sup.6                       2'-O-Me/RNA.sup.c                                                                       0.1       37       5 × 10.sup.-11                                                                  2 × 10.sup.5                       DNA/RNA.sup.c                                                                           0.1       37       2 × 10.sup.-9                                                                   --                                       DNA/DNA.sup.d                                                                           0.2       39       --      3 × 10.sup.5                       ______________________________________                                         .sup.a The buffer was 100 mM NaCl, 10 mM phospphate, 1 mM EDTA, pH 7.0,       and the dissociation constant was determined from gel-shift experiments       using a .sup.32 P-labeled RNA. The rate was measured in experiments which     were stopped by adding 50-fold excess of unlabeled RNA followed by rapid      freezing.                                                                     .sup.b The rate constants were determined from plots of the pseudo-first      order rate constant versus the reagent concentration, which in all cases      were much higher than the concentration of the labeled RNA target.            .sup.c The sequence of the reagent was: N-(or                                 5')-TGTACGTCACAACTA-C-terminal (or 3'), and the sequence of the RNA           target: 5'-TAGTTGTGACGTACA-3. 2'-O-Me is the 2'-O-methyl RNA derivative.      .sup.d Results taken from Tibanyenda et. al., Eur. J. Biochem. 1984, 139,     19. The sequence was: 5'-CAACTTGATATTAATA.                               

The following chemical synthesis are shown in Schemes I and II.

Scheme I

EXAMPLE 36

KonoBoc-Diethylenetriamine Dihydrochloride (1)

A solution of t-butyl-p-nitrophenyl carbonate (10 g; 0.0418 mole) inCHCl₃ (400 ml) was added to a solution of diethylenetriamine (45 ml;0.0417 mole) in CHCl₃ (250 ml) at 0° C. over a period of 3 h. Thereaction mixture was stirred overnight at room temperature. Theprecipitate that appeared was filtered and washed in CHCl₃. The solventwas evaporated, first under reduced pressure with a water-aspirator,then with an oil-pump (0.05 mmHg; 50° C). The residue was dissolved in amixture ethylacetate (50 ml)/H₂ O (50 ml) and the solution was acidifiedto pH 4 with HCl 4N, extracted with ethylacetate (3×50 ml). The aqueoussolution was adjusted to pH 9 with NaOH 2N and extracted withethylacetate (3×50 ml). The aqueous phase was adjusted to pH 11.5 andextracted with ethylacetate (10×50 ml). The combined organic phases ofthe last extraction were evaporated under reduced pressure and theresulting oil was dissolved in water (50 ml) and acidified to pH 5.Evaporation of water yielded a slightly yellow solid, which wasthoroughly washed with ether (yield: 6.41 g; 55%). ¹ H-NMR (D₂ O): δ(ppm): 1.4 (s, 9H); 3.0 (t, 2H); 3.3 (s broad, 4H); 3.4 (t, 2H)

EXAMPLE 37

Boc-, Z-diethylenetriamine Hydrochloride (2)

To a solution of 1 (5.5 g; 19.9 mmoles) in dioxane (50 ml)/water (50 ml)adjusted at pH 11 was added a solution of benzylnitrophenyl carbonate(5.44 g; 19.9 moles) in dioxane (50 ml) at 0° C. over a period of lh,while maintaining the pH at 11 with NaOH 2N. The reaction mixture wasthen stirred at room temperature for 1.5 h. Subsequently, ethylacetate(100 ml) was added and the reaction mixture was cooled at 0° C. andacidified to pH 4 with HCl 4N. A precipitate appeared in the organicphase. The two phases were separated and the organic phase filtered.This (addition of ethylacetate, separating the two phases and filteringthe organic phase) was repeated 3 times to give 3.05 g of white solid.An additional 1.38 g was obtained by joining all the organic phases andadding ether (Yield: 4.427 g; 59%). ¹ H-NMR (DMSO d6): δ (ppm): 1.5 (s,9H); 3.0 (m, 4H); 3.4 (m, 4H); 5.1 (s, 2H); 7.4 (s, 5H); 7.1 (m, 1H);7.6 (m, 1H). MS FAB+: M+1: 338.2

EXAMPLE 38

Coupling of 2 with N-1-Carboxymethylthymine

Compound 2 (4 g; 10.7 mmoles), N-1-carboxymethylthymine (1.97 g; 10.7mmoles), DhbtOH (1.75 g; 10.7 mmoles) and DIEA (1.2 ml; 12 mmoles) weredissolved in a mixture of CH₂ Cl₂ (50 ml)/DMF (50 ml). After cooling at0° C., DCU (2.2 g; 10.7 mmoles) was added and the reaction mixture wasstirred at 0° C. for 1/2 h and 2.5 h at room temperature. DCU wasfiltered and washed with CH₂ Cl₂ (2×100 ml). The combined organic phaseswere washed with NaHCO₃ 1M (3×100 ml), KHSO₄ 1M (3×100 ml), H₂ O (2×100ml), dried over Na₂ SO₄, filtered and evaporated under reduced pressure.The resulting oil was crystallized in chloroform/ether to yield 4.04 g(75%) of product. ¹ H-NMR (CDCl₃); δ (ppm); 1.4 (s, 9H); 1.9 (s, 3H);3.4 (m, 8H); 4.4 (d, 2H); 5.1 (d, 2H); 5.5 (m, 1H); 5.9 (m, 1H); 6.9 (s,1H); 7.4 (s, 5H); 9.0 (s, 1H). MSFAB+: M+1; 504.

EXAMPLE 39

Hydrogenolysis of 3 to 4

Compound 3 (4 g; 7.9 mmoles) was dissolved in MeOH (150 ml). At 0° C.,10% Pd/C (1.4 g) was added. The reaction mixture was hydrogenated at 0°C. for 1 h, filtered through Celite and the solvent removed underreduced pressure to give 3.0 g (100%) of 4. ¹ H-NMR (CDCl₃); δ (ppm);1.4 (s, 9H); 1.9 (s, 3H); 3.0-3.8 (m); 4.6 (d, 2H); 6,9 (s, 1H); 7.3 (s,5H); 9.0 (s, 1H). MSFAB+:M+1: 370.2

EXAMPLE 40

N-(benzyl acetate)-Glycine Ethyl Ester Hydrochloride (5)

At room temperature, benzyl bromo acetate (5.7 ml; 36 mmoles) was addedover a period of 10 min. to a solution of glycine ethyl esterhydrochloride (5 g; 36 mmoles) and triethylamine (10.43 ml; 0.072 moles)dissolved in absolute EtOH (100 ml). After 4 days at room temperature,the solvent was removed under reduced pressure. The residue wasdissolved in ethylacetate(50 ml)/H₂ O) (25 ml). After separation of thetwo phases, the organic phase was thoroughly washed with water (8×25ml). After evaporation of the solvent, the resulting oil was dissolvedin ether (20 ml)/water (30 ml) and acidified to pH 4.5. After separationof the two phases, the aqueous phase was concentrated and the resultingoil crystallized in cold ether to yield 4 g (39%) of the product. ¹H-NMR (CDCl₃); δ (ppm); 1.3 (t, 3H); 4.1 (d, 4H); 4.4 (q, 2H); 5.3 (d,2H); 5.5 (m, 1H); 7.4 (s, 5H). MS FAB+; M+1: 252.1

EXAMPLE 41

Coupling of 5 with N-1-Carboxymethylthymine to give 6

Compound 5 (2.8 g; 9.7 mmoles), N-1-carboxymethylthymine (1.79 g; 9.7mmoles), DhbtOH (1.6 g; 9.7 mmoles) and DIEA (1.7 ml; 10 mmoles) weredissolved in a mixture of CH₂ Cl₂ (30 ml)/DMF (30 ml). After cooling at0° C., DCC (2.0 g; 9.7 mmoles) was added and the reaction mixture wasstirred at 0° C. for 1/2 h and 4 h at room temperature. The DCU wasfiltered and washed with CH₂ Cl₂ (120 ml). The combined organic phaseswere washed with NaHCO₃ 1M (3×60 ml), KHSO₄ 1M (3×60 ml), H₂ O (2×60ml), dried over Na₂ SO₄, filtered and evaporated under reduced pressure.The resulting oil was crystallized in chloroform (60 ml)/petroleum ether(120 ml) to yield 3.235 g (80%) of 6. ¹ H-NMR (DMSO d6): δ (ppm); 1.3(dt, 3H); 1.9 (s, 3H); 4.2 (m, 4H)); 4.4 (d, 2H); 5.3 (d, 2H); 7.2 (s,1H); 7.4 (s, 5H); 11.3 (s, 1H). MS FAB+: M+1: 418.1

EXAMPLE 42

Hydrogenolysis of 6 to 7

Compound 6 (4 g; 9.6 mmoles) was dissolved in MeOH (100ml)/DMF (25 ml).At 0° C., 10% Pd/C (1.6 g) was added. The reaction mixture washydrogenated at 0° C. for 1H, filtered through Celite and the solventremoved under reduced pressure to give 3.12 g (99%) of 7. ¹ H-NMR (DMSOd6): δ (ppm); 1.3 (dt, 3H1); 1.9 (s, 3H); 4.0-4.5 (m); 4.9 (s, 2H); 7.2(s, 1H); 11.3 (s, 1H). MS FAB+: M+1: 328.1

EXAMPLE 43

Coupling of 4 and 7 to Give 8

Compound 4 (2.8 g; 7.6 mmoles), 7 (2.5 g; 7.6 mmoles), DhbtOH (1.24 g;7.6 mmoles) were dissolved in a mixture of CH₂ Cl₂ (50 ml)/DMF (50 ml).After cooling at 0° C., DCC (1.56 g; 7.6 mmoles) was added and thereaction mixture was stirred at 0° C. for 1/2 h and overnight at roomtemperature. The DCU was filtered and washed with CH₂ Cl₂ (250 ml). Thecombined organic phases were washed with NaHCO₃ 1M (3×100 ml), KHSO₄ 1M(3×100 ml), H₂ O (2×100 ml), dried over Na₂ SO₄, filtered and evaporatedunder reduce pressure. The resulting oil was crystallized in chloroform(30 ml)/petroleum ether (60 ml). This material was recrystallized inchloroform (30 ml)/petroleum ether (25 ml) to give 0.608 g (12%) of 8.MS FAB+: M+1: 418.1

EXAMPLE 44

Hydrolysis of 8 to Give 9

Compound 8 (25 mg, 0.037 mmoles) was dissolved in absolute ethanol (5ml). Then NaOH 1M (1 ml) was added. The reaction mixture was stirred for35 min. at room temperature and after cooling, neutralized with Dowex 50W (ca. 1 g). After filtration and evaporation of the solvent, the acid 9was obtained as a white solid, yield 18 mg (82%).

Scheme II

EXAMPLE 45

Alanine benzyl ester (10)

Thionylchloride (9.8 ml, 135 mmol, 1.2 eqv) was added dropwise during 15min. to benzyl alcohol (210 ml) stirred under nitrogen and cooled to-10° C. Alanine (10.0 g, 112 mmol, 1.0 eqv) was added in portions during10 min. The reaction mixture was heated overnight at 60-70° C. Thereaction mixture was evaporated in vacuo and the residue was dissolvedin water (150 ml). The pH was adjusted to 1-2 by addition of 4N HCl(aq.) and the aqueous phase was extracted with dichloromethane (2×200ml). The organic phase was washed with hydrochloric acid (pH 1, 1×50ml). The aqueous phases were collected, alkalinized (to pH 9-10) byaddition of 2N NaOH (aq.) and subsequently extracted withdichloromethane (2×200 ml and 1×100 ml--alkaline extract). The alkalineextract was dried (Na₂ SO₄), filtered and evaporated under reducedpressure affording 13.39 g (67%) of the title compound as a colorlessoil. The product was characterized as its hydrochloride: The product(0.50 g, 2.8 mmol) was dissolved in ether (5 ml) and HCl in ether (2 ml)was added. The precipitate was collected by filtration and washed withether (2×5 ml) yielding 0.57 g (95%) of white crystalline alanine benzylester hydrochloride. ¹ H-NMR (D₂ O/TMS): δ 1.48 (unresolved d, 3H, Me);4.15 (unresolved q, 1H, CH); 5.20 (s, 2H, CH₂); 7.38 (br s, 5H, Ph). ¹³C-NMR (D₂ O/TMS): δ 14.7 (Me); 48.4 (CH); 68.1 (CH₂); 128.1, 128.5,134.3 (Ph); 170.1 (C═O). MS(FAB+) m/z (%): 180 (100, M-HCl+H). Anal.Calcd. for C₁₀ H₁₄ ClNO₂ : C, 55.69; H, 6.54; N, 6.49. Found: C, 55.60;H, 6.56; N, 6.40.

EXAMPLE 46

N-(2-Boc-aminoethyl) Alanine Benzyl Ester Hydrochloride (11)

Alanine benzyl ester (10, 8.82 g, 49 mmol, 1 eq) was dissolved in MeOH(100 ml) and glacial acetic acid (10.34 g, 172 mmol, 3.5 eq) was added.The mixture was stirred at 0° C. under nitrogen and sodiumcyanoborohydride (10.82 g, 172 mmol, 3.5 eq) was added.Boc-aminoacetaldehyde (15.67 g, 98 mmol, 2 eq) in MeOH (200 ml) wasadded dropwise during 2 h. The reaction mixture was stirred at 0° C. for35 min. and then at 4-5° C. overnight. Water (300 ml) was added to thereaction mixture and the pH was adjusted to 9 by addition of solidsodium carbonate. The saturated solution was filtered and subsequentlyextracted with ether (3×500 ml). The organic phase was washed with a 1:1mixture of saturated aqueous solutions of sodium chloride and sodiumbicarbonate (1×450 ml), dried (Na₂ SO₄), filtered and evaporated underreduced pressure to give 22.60 g of crude N-(2-Boc-amino-ethyl)alaninebenzyl ester as a slightly golden oil. The crude product was dissolvedin dry ether (500 ml) and stirred at 0° C. HCl in ether (60 ml) was thenadded to the solution, and the resulting precipitate was collected byfiltration and washed with cold dry ether (3×20 ml) affording 7.21 g(41%) of 11 as white crystals. A second crop of 2.12 g (12%) could becollected as a white semicrystalline material by keeping the motherliquor in the freezer overnight. This second crop was slightly less purethan the first crop. ¹ H-NMR (D₂ O/TMS): δ 1.36 (s, 9H, Boc); 1.52 (d,J=7.2 Hz, 3H, Me); 3.16 (m, 2H, NCH₂); 3.35 (m, 2H, NCH₂); 4.17 (q,J=7.2 Hz, 1H, CH); 5.26 (s, 2H, CH₂ Ph); 7.40 (s, 5H, Ph). ¹³ C-NMR (D₂O/TMS): δ 13.5 (Me); 27.3 (Box); 36.4 (CH); 45.5 (NCH₂); 55.2 (NCH₂);68.4 (CH₂ Ph); 128.3, 128.7, 128.8 (Ph). MS(FAB+) m/z (%): 323 (100,M-HCl+H) Anal. Calcd for C₁₇ H₂₇ CIN₂ O₄ : C, 56.90; H, 7.58; N, 7.81;Cl, 9.88. Found: C, 55.11; H, 7.52; N, 7.93; Cl, 10.45.

EXAMPLE 47

N-(2-Boc-Aminoethyl)-N-(1-Thyminylacetyl)Alanine Benzyl Ester (12)

N-(2-Boc-aminoethyl)alanine benzyl ester hydrochloride (11, 3.00 g, 8.4mmol, 1.0 eq) was dissolved in dry dichloromethane (50 ml) and stirredat 0° C. under nitrogen. DIEA (1.5 ml, 8.4 mmol, 1.0 eq), DHBtOH (1.50g, 9.2 mmol, 1.1 eq) in dichloromethane (30 ml), 1-thyminylacetic acid(1.69 g, 9.2 mmol, 1.1 eq) in dichloromethane (30 ml) and DCC (2.07 g,10.0 mmol, 1.2 eq) in dichloromethane (40 ml) were added in that order.The reaction mixture was stirred at 0° C. for 1 h and then at 4-5° C.overnight. The precipitated DCU was filtered off and washed withdichloromethane (3×15 ml). The filtrate was washed with saturatedaqueous sodium bicarbonate (3×150 ml) diluted to five times its volume,saturated aqueous potassium hydrogen sulfate (2×150 ml) diluted to threetimes its volume, saturated aqueous sodium chloride (1×150 ml), dried(Na₂ SO₄), filtered and finally evaporated under reduced pressureyielding 3.83 g of crude product as a pink glassy foam. The crudeproduct was chromatographed (silica, 0.063-0.200 mm) usingMeOH/dichloromethane as the eluent (3/97, v/v until the first fractionappeared then 5/95, v/v) affording 2.87 g (70%) of 12 a white glassyfoam. 12 was isolated as mixture of two conformers due to restrictedrotation around the amide bond. Consequently, some of the signals in theNMR spectra were split into a major and minor component. The valuesprovided below are for the major component. ¹ H-NMR (CDCl₃ /TMS): δ 1.44(s, 9H, Boc); 1.52 (d, J=7.1 Hz, 3H, ala-Me); 3.31 (m, 2H, NCH₂); 3.44(m, 1H, NCH₂); 3.55 (m, 1H, NCH₂); 4.34 (q, J=7.3 Hz, 1H, CH); 4.53 (s,2H, acetyl--CH₂); 5.16 (s, 2H, CH₂ Ph); 6.90 (s, 1H, thymine-H-6); 7.35(m, 5H, Ph). ¹³ C-NMR (CDCl₃ /TMS): δ 12.2 (thymine-Me); 14.3 (ala-Me);28.3 (Boc); 39.3 (CH); 46.0 (acetyl-CH₂); 48.0 (NCH₂); 55.6 (NCH₂); 67.2(CH2 Ph); 79.8 (Boc); 110.5 (thymine-C-5); 128.0, 128.3, 128.5, 128.6,135.1 (Ph); 140.8 (thymine-C-6); 150.9 (thymine-C-2); 155.8 (Boc-C═O);164.1 (thymine-C-4); 166.8 (acetyl-CH₂); 171.3 (ester-C═O). MS(FAB+) m/z(%): 489 (64, M+H). Anal. Calcd for C₂₄ H₃₂ N₄ O₇ : C, 59.01; H, 6.60;N, 11.47. Found: C, 58.55; H, 6.65; N, 11.16.

EXAMPLE 48

N-(2-Boc-Aminoethyl)-N-(1-Thyminylacetyl)Alanine (13)

N-(2-Boc-aminoethyl)-N-(1-thyminylacetyl)alanine benzyl ester (12, 2.02g, 4.1 mmol, 1 eq) was dissolved in MeOH (100 ml) and stirred at 0° C.under nitrogen. 10% palladium on activated carbon (1.7 g) was added andthe mixture was hydrogenated at atmospheric pressure and 0° C. for 1 hat which time hydrogen uptake had ceased (91 ml, 4.1 mmol, 1 eq had beenconsumed). The reaction mixture was filtered through celite which waswashed thoroughly with MeOH. The collected filtrates were evaporatedgiving 1.65 g (100%) of 13 as a white glassy foam. 13 was isolated as amixture of two conformers due to restricted rotation around the amidebond. Consequently, some of the signals in the NMR spectra were splitinto a major and minor component. The values provided below are for themajor component. ¹ H-NMR (DMSO-d₆ /TMS): δ 1.42 (d, J=7.1 Hz, 3H,ala-Me); 1.46 (s, 9H, Boc); 3.26 (m, 2H, NCH₂); 3.42 (m, 2H, NCH₂); 4.39(q, J=7.0 Hz, 1H, CH); 4.69 (s, 2H, acetyl-CH₂); 7.00 (s, 1H,thymine-H-6); 11.35 (s, 1H, COOH). ¹³ C-NMR (DMSO-d₆ /TMS): δ 12.0(thymine-Me); 14.8 (ala-Me); 28.3 (Boc); 33.6 (CH); 45.7 (acetyl-CH₂);48.0 (NCH₂); 54.8 (NCH₂); 78.1 (Boc); 108.1 (thymine-C-5); 142.2(thymine-C-6); 151.0 (thymine-C-2); 155.8 (Boc-C═O); 164.4(thymine-C-4); 166.9 (acetyl--CH₂); 172.6 (acid-C═O). MS(FAB-) m/z (%):397 (100, M-H); 398 (21, M-H+1); 399 (5, M-H+2). Anal. Calcd for C₁₇ H₂₆N₄ O₇ : C, 51.25; H, 6.58; N, 14.06.

EXAMPLE 49

Chimera Macromolecule Having Peptide Nucleic Acids Section Attaching to3' Terminus of a 2'-Deoxy Phosphorothioate Oligonucleotide Section

A first section of peptide nucleic acids is prepared as per PCT patentapplication PCT/EP/01219. The peptide nucleic acids are prepared fromthe C. terminus towards the N terminus using monomers having protectedamine groups. Following completion of the peptide region, the terminalamine blocking group is removed and the resulting amine reacted with a3'-C-(formyl)-2',3'-dideoxy-5'-trityl nucleotide as prepared as per theprocedure of Vasseur, et. al., J. Am. Chem. Soc. 1992, 114, 4006. Thecondensation of the amine with the aldehyde moiety of the C-formylnucleoside is effected as per the conditions of the Vasseur, ibid., toyield an intermediate oxime linkage. The oxime linkage is reduced underreductive alkylation conditions of Vasseur, ibid., with HCHO/NaBH₃CN/AcOH to yield the nucleoside connected to the peptide nucleic acidvia an methyl alkylated amine linkage. An internal 2'-deoxyphosphorothioate nucleotide region is then continued from thisnucleoside as per standard automatated DNA synthetic protocols (seeOligonucleotide synthesis, a practic approach, M. J. Gait ed, IRL Press,1984).

EXAMPLE 50

Chimera Macromolecule Having Peptide Nucleic Acids Section Attaching to5' Terminus of a Phosphorothioate Oligonucleotide Section

A phosphorothioate oligonucleotide is prepare in the standard manner ona solid support as per standard protocols (see Oligonucleotides andAnalogues, A Practical Approach, F. Eckstein Ed., IRL Press, 1991. Thedimethoxytrityl blocking group on that nucleotide is removed in thestandard manner. Peptide synthesis for the peptide region is commencedby reaction of the carboxyl end of the first peptide nucleic acid ofthis region with the 5' hydroxy of the last nucleotide of the DNAregion. Coupling is effected via DEA in pyridine to form an esterlinkage between the peptide and the nucleoside. Peptide synthesis isthen continued in the manner of patent application PCT/EP/01219 tocomplete the peptide nucleic acid region.

EXAMPLE 51

Double-stranded Duplex Structures That Include Chimera Stand andTriple-stranded Triplex structures That Include Chimera Strand

Duplex and triplex structures will be formed with the chimera strands ofExamples 42 and 43 as per the protocols of other of the above examples.Duplex structures can include duplexes between a PNA-RNA or PNA-DNAstrand and a RNA strand, a PNA-RNA or PNA-DNA strand and a DNA strand, aPNA-RNA or PNA-DNA strand and a PNA strand or a PNA-RNA or PNA-DNAstrand and a further chimeric PNA-DNA or PNA-RNA strand. Triplexstructures can include a PNA containing chimeric strands triplexing withdsDNA or with a double-stranded PNA construct. Further triplexstructures can include two of the PNA containing chimera triplexing witha single DNA or RNA strand. Additional triplex structures can include asingle PNA containing chimera plus a PNA triplexing with an additionalPNA containing chimera, an additional PNA strand or a DNA or RNA strand.

EXAMPLE 52

Binding Between Single Strand PNA Containing Chimera and TranscriptionFactor or other Protein

A PNA containing chimeric strand will be used to bind to or otherwisemodulate single-stranded DNA, double-stranded DNA, RNA, a transcriptionfactor or other protein. In use of a PNA containing chimera, part of thebinding between the chimera and the transcription factor or otherprotein will be binding between the sugar-phosphate backbone of the DNAor RNA portion of the chimera and hydrogen bonding between the ligands,e.g., nucleobases, of the PNA portion of the chimera. Binding to thesugar-phosphate backbone includes binding to phosphodiester linkages,phosphorothioate linkages or other linkgages that may be used as thebacbone of the DNA or RNA. In other instances, bonding can includehydrophobic contacts between hydrophobic groups on the ligands,including nucleobases, of the PNA or the nucleobases of the nucleic acidportion of the chimera with like hydrophobic groups on proteins that arebeing bound. Such hydrophobic groups on the chimeric strand include themethyl groups on thymine nucleobases.

EXAMPLE 53

PNA Dimer (detT-idaT) Having "Reversed" Monomer ##STR4##

A. MonoBoc-diethylenetriamine dihydrochloride.

A solution of t-butyl-p-nitrophenyl carbonate (10 g; 0.0418 mole) inCHCl₃ (400 ml) was added to a solution of diethylenetriamine (45 ml;0.417 mole) in CHCl₃ (250 ml) at 0° C. over a period of 3 h. Then, thereaction mixture was stirred overnight at room temperature.

The precipitate that appeared was filtered and washed with CHCl₃. Thesolvent was evaporated, first under reduced pressure with awater-aspirator, then with an oil-pump (0.05 mmHg; 50° C.).

The residue was dissolved in a mixture of ethyl acetate (50ml) and H₂ O(50 ml). The solution was acidified to pH 4 with 4N HCl and extractedwith ethyl acetate (3×50 ml). The aqueous solution was adjusted to pH 9with 2N NaOH and extracted with ethyl acetate (3×50 ml). Then, theaqueous phase was adjusted to pH 11.5 and extracted with ethyl acetate(10×50 ml). The combined organic phases of the last extraction wereevaporated under reduced pressure and the resulting oil was dissolved inwater (50 ml) and acidified to pH 5. Evaporation of water yielded aslightly yellow solid, which was thoroughly washed with ether (yield:6.41 g; 55%).

¹ H-NMR (D₂ O): δ (ppm): 1.4 (s,9H); 3.0 (t,2H); 3.3 (s broad, 4H); 3.4(t, 2H)

B. Boc-, Z-diethylenetriamine hydrochloride.

To a solution containing the product of Example 53A (5.5 g: 19.9 mmoles)in dioxane (50 ml)/water (50 ml) adjusted at pH 11 was added a solutionof benzyl-nitrophenyl carbonate (5.44 g; 19.9 mmoles) in dioxane (50 ml)at 0° C. over a period of 70 min, while maintaining the pH at 11 with 2NNaOH. The reaction mixture was then stirred at room temperature for 1.5h.

Subsequently, ethyl acetate (100 ml) was added and the reaction mixturewas cooled at 0° C. and acidified to pH 4 with 4N HCl. A precipitateappeared in the organic phase. The two phases were separated and theorganic phase filtered.

These operations (adding ethyl acetate, separating the two phases andfiltering the organic phase) were repeated 3 times. A white solid (3.05g) was collected this way. An additional 1.38 g was obtained by joiningall the organic phases and adding ether. (Yield : 4.427 g; 59%)

¹ H-NMR (DMSO d6): δ (ppm): 1.5 (s,9H); 3.0 (m,4H); 3.4 (m,4H); 5.1(s,2H); 7.4 (s,5H); 7.1 (m,lH); 7.6 (m,1H). MS FAB+: M+1: 338.2

C. Coupling with N-1-carboxymethylthymine.

The product of Example 53B (4g; 10.7 mmoles), N-1-carboxymethylthymine(1.97 g; 10.7 mmoles). DhbtOH (1.75 g; 10.7 mmoles) and DIEA (1.9 ml;11.2 mmoles) were dissolved in a mixture of CH₂ Cl₂ (50 ml)/DMF (50 ml).After cooling at 0° C., DCC (2.2 g; 10.7 mmoles) was added and thereaction mixture was stirred at 0° C. for 0.5 h and 2.5 h at roomtemperature. Then, DCU was filtered and washed with CH₂ Cl₂ (2×100 ml).The combined organic phases were washed with 1M NaHCO₃ (3×100 ml), 1MKHSO₄ (3×100 ml), H₂ O (2×100 ml), dried over Na₂ SO₄, filtered andevaporated under reduced pressure. The resulting oil was crystallized inchloroform/ether. (Yield : 4.04 g; 75%)

¹ H-NMR (CDCl₃): δ (ppm): 1.4 (s,9H); 1.9 (s,3H); 3.4(m, 8H); 4.4(d,2H); 5.1 (d,2H): 5.5 (m,lH); 5.9 (m,1H); 6.9 (s,1H): 7.4 (s,5H); 9.0(s,1H). MS FAB+: M+1: 504

D. Hydrogenolysis

The product of Example 53C. (4 g; 7.9 mmoles) was dissolved in MeOH (150ml). At 0° C., 10% Pd/C (1.4 g) was added. The reaction mixture washydrogenated at 0C. for 1 h, filtered through Celite and the solventremoved under reduced pressure to give 4. (Yield: 3,0 g; 100%)

¹ H-NMR (CDCl₃): δ (ppm): 1.4 (s,9H); 1.9 (s,3H); 3.0-3.8(m); 4.6(d,2H); 6.9 (s,1H); 7.3 (s,5H); 9.0 (s,1H). MS FAB+: M+1: 370.2

E. N-(benzyl acetate)-glycine ethyl ester hydrochloride.

At room temperature, benzyl bromoacetate (5.7 ml; 36 mmoles) was addedover a period of 10 min to a solution of glycine ethyl esterhydrochloride (5 g; 36 mmoles) and triethylamine (10.43 ml; 0.072 moles)dissolved in absolute ethanol (100 ml). After 4 days at roomtemperature, the solvent was removed under reduced pressure. The residuewas dissolved in ethyl acetate (50 ml)/H₂ O (25 ml). After separation ofthe two phases, the organic phase was thoroughly washed with water (8.25ml). After evaporation of the solvent, the resulting oil was dissolvedin ether (20 ml)/water (30 ml) and acidified to pH 4.5. After separationof the two phases, the aqueous phase was concentrated and the resultingoil crystallized in cold ether. (Yield 4 g; 39%)

¹ H-NMR (CDCl₃): δ (ppm): 1.3 (t,3H); 4.1 (d,4H); 4.4 (q,2H); 5.3(d,2H); 5.5 (m,1H); 7.4 (s,5H). MS FAB+: M+1: 252.1

E. Coupling N-1-carboxymethylthymine.

The product of Example 53D (2.8 g; 9.7 mmoles), N-1-carboxymethylthymine(1.79 g; 9.7 mmoles), DhbtOH (1.6 g; 9.7 mmoles) and DIEA (1.7 ml; 10mmoles) were dissolved in a mixture of CH₂ Cl₂ (30 ml)/DMF (30 ml).After cooling at 0° C., DCC (2.0 g; 9.7 mmoles) was added and thereaction mixture was stirred at 0° C. for 0.5 h and 4 h at roomtemperature. Then DCU was filtered and wash with CH₂ Cl₂ (120 ml). Thecombined organic phases were washed with 1M NaHCO₃ (3×60 ml), 1M KHSO₄(3×60 ml), H₂ O (2×60 ml), dried over Na₂ SO₄, filtered and evaporatedunder reduced pressure. The resulting oil was crystallized in chloroform(60 ml)/petroleum ether (120 ml). (Yield: 3.235 g; 80%)

¹ H-NMR (DMSO d6): δ (ppm): 1.3 (dt,3H); 1.9 (s,3H); 4.2 (m,4H); 4.4(d,2H); 5.3 (d,2H); 7.4 (s,1H); 7.4 (s, 5H); 11.3 (s,1H). MS FAB+: M+1:418.1

F. Hydrogenolysis

The product of Example 53E (4g; 9.6 mmoles) was dissolved in methanol(lOOml)/DMF (25 ml). At 0° C., 10% Pd/C (1.6 g) was added. The reactionmixture was hydrogenated at 0° C. for 1 h, filtered through Celite andthe solvent removed under reduced pressure. (Yield=3.12 g; 99%)

¹ H-NMR (DMSO d6): δ (ppm): 1.3 (dt,3H); 1.9 (s,3H); 4.0-4.5 (m); 4.9(s,2H); 7.2 (s,1H); 11.3 (s,1H). MS FAB+: M+1: 328.1

G. coupling

The products of Examples 53D (2.8 g; 7.6 mmoles) and 53F (2.5 g; 7.6mmoles), and DhbtOH (1.24 g; 7.6 mmoles) were dissolved in a mixture ina mixture of CH₂ Cl₂ (50 ml)/DMF (50 ml). After cooling at 0° C., DCC(1.56g; 7.6 mmoles) was added and the reaction mixture was stirred at 0°C. for 0.5 h and overnight at room temperature. Then, DCU was filteredand washed with CH₂ Cl₂ (250 ml). The combined organic phases werewashed with 1M NaHCO₃ (3×100 ml), 1M KHSO₄ (3×100 ml), H20 (2×100 ml),dried over Na₂ SO₄, filtered and evaporated under reduced pressure. Theresulting oil was crystallized in chloroform (30 ml)/petroleum ether (60ml). This material was recrystallized in chloroform (30 ml)/petroleumether (25 ml) to give a pure compound. (Yield: 0.60 g; 12%)

MS FAB+: M+1: 418.1

H. Hydrolysis

The product of Example 53G (529 mg; 0.8 mmoles) was dissolved inabsolute ethanol (100 ml). Then NaOH 1M (20 ml) was added. The reactionmixture was stirred for 30 min at room temperature and after cooling,neutralized with Dowex 50 W (about 13 g). After filtration, washing withH₂ O, ethanol and evaporation of the solvent, the residue was suspendedin ether and filtered. The acid product was obtained as a white solid,pure in HPLC (Yield: 450 mg: 86%)

MS FAB+: 673 (M+Na⁺); 617 (M Na⁺ -tBu); 595 (M+1-tBu); 573 (M Na⁺ -Boc);551 (M+1-Boc)

EXAMPLE 54

PNA Oligomer Having "Reversed" Monomer

The dimer prepared in Example 53 was incorporated into PNAH-TT(detT-idaT)CCTCTC-LysNH₂ (SEQ ID NO:53) generally in accordance withExample 1. The melting temperatures, T_(m), of complexes between thisPNA and decamers 5'd(AAAAGGAGAG) (SEQ ID NO:54) and 5'd(GAGAGGAAAA) (SEQID NO:55) were 55° C. and 43.5° C., respectively, at pH 7. Bycomparision, the T_(m) of complexes between PNA H-TTTCCTCTC-LysNH₂ (SEQID NO:56), prepared generally in accordance with Example 1, and thesedecamers was 58.5° C. and 40.5° C., respectively, at pH 7.

Those skilled in the art will appreciate that numerous changes andmodifications may be made to the preferred embodiments of the inventionand that such changes and modifications may be made without departingfrom the spirit of the invention. It is therefore intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 56                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 11                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ly - #s                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 11                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ly - #s                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 10                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #        10                                                                   - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 10                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #        10                                                                   - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 9                                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #          9                                                                  - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 11                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ly - #s                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 7                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Xaa Xaa Xaa Xaa Xaa Xaa Lys                                                 1               5                                                             - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 11                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ly - #s                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 11                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ly - #s                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 15                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 12                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 13                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 15                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa             #                15                                                           - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 9                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 9                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 15                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 12                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 13                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 15                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa             #                15                                                           - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 15                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 12                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 13                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 15                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa             #                15                                                           - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 9                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 9                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 16                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 12                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 13                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 15                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 16                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa         #                15                                                           - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 16                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 12                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 13                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 15                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 16                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa         #                15                                                           - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 10                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 10                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 15                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 12                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 13                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 15                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa             #                15                                                           - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 29                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 12                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 13                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 15                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 16                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 18                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 19                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 20                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa             #                15                                                           - Xaa Xaa Xaa Xaa Xaa Lys Lys Lys Lys Lys Ly - #s Lys Lys Lys                 #                25                                                           - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 21                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 12                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 13                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 15                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 16                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 18                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 19                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 20                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa             #                15                                                           - Xaa Xaa Xaa Xaa Xaa Lys                                                                     20                                                            - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 12                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 13                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 15                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 16                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 18                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 19                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 20                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa             #                15                                                           - Xaa Xaa Xaa Xaa Xaa                                                                         20                                                            - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 21                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 12                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 13                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 15                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 16                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 18                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 19                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 20                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa             #                15                                                           - Xaa Xaa Xaa Xaa Xaa Lys                                                                     20                                                            - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 12                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 13                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 15                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 16                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 18                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 19                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 20                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa             #                15                                                           - Xaa Xaa Xaa Xaa Xaa                                                                         20                                                            - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 21                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 12                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 13                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 14                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 15                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 16                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 18                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 19                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 20                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa             #                15                                                           - Xaa Xaa Xaa Xaa Xaa Lys                                                                     20                                                            - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 12                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Beat isoform of alanine."                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                - Xaa Xaa Xaa Xaa Ala Xaa Xaa Xaa Xaa Xaa Xa - #a Lys                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 12                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Beat isoform of alanine."                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 11                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                - Xaa Xaa Xaa Xaa Ala Xaa Xaa Xaa Xaa Xaa Xa - #a Lys                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 26                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                #              26  AAAA AGAAAA                                                - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                # 20               TTTT                                                       - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 38                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                #     38           GTCG ACTTTTCTTT TTGTCGAC                                   - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                # 20               TTTT                                                       - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 16                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                #    16                                                                       - (2) INFORMATION FOR SEQ ID NO:35:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 16                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                #    16                                                                       - (2) INFORMATION FOR SEQ ID NO:36:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 16                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                #    16                                                                       - (2) INFORMATION FOR SEQ ID NO:37:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 16                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                #    16                                                                       - (2) INFORMATION FOR SEQ ID NO:38:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 16                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                #    16                                                                       - (2) INFORMATION FOR SEQ ID NO:39:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 16                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                #    16                                                                       - (2) INFORMATION FOR SEQ ID NO:40:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 9                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:41:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 5                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                - Xaa Xaa Xaa Xaa Xaa                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:42:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 98                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                #              50AAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA                      #                98AAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAA                        - (2) INFORMATION FOR SEQ ID NO:43:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 10                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                #        10                                                                   - (2) INFORMATION FOR SEQ ID NO:44:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 10                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                #        10                                                                   - (2) INFORMATION FOR SEQ ID NO:45:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 8                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:46:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 50                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                #              50ACAGCA TATTTTCTTT TAAAATTAGC AGGAAGATGG                      - (2) INFORMATION FOR SEQ ID NO:47:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 15                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:48:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 11                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ly - #s                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:49:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 11                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ly - #s                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:50:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 16                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                #    16                                                                       - (2) INFORMATION FOR SEQ ID NO:51:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 8                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 1 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:52:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 15                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:53:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 11                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ly - #s                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:54:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 10                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                #        10                                                                   - (2) INFORMATION FOR SEQ ID NO:55:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 10                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                #        10                                                                   - (2) INFORMATION FOR SEQ ID NO:56:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 11                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/label= MODIFIED-SITEFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 2                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 5                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 7                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/label= Modified-siteFORMATION:                                              #"Heterocyclic base is attached to                                                           N-acetyl(2-a - #minoethyl)glycine through the N-acetyl         #position 9 of the base."                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ly - #s                             #                10                                                           __________________________________________________________________________

We claim:
 1. A complex comprising a first nucleic acid strand and secondand third strands, wherein:said second and third strands independentlycontain a sequence of liqands covalently bound by linking moieties; aplurality of said ligands on said second strand interact with said firststrand; a plurality of said ligands on said third strand interact withsaid first strand or with said ligands on said second strand; and saidsecond strand and said third strand, independently, comprise a compoundof the formula III, IV or V: ##STR5## wherein: each L is independentlyselected from the group consisting of hydrogen, phenyl, heterocyclicmoieties, naturally occurring nucleobases, and non-naturally occurringnucleobases;each R^(7') is independently selected from the groupconsisting of hydrogen and the side chains of naturally occurring alphaamino acids; n is an integer greater than 1, each k, l, and m is,independently, zero or an integer from 1 to 5; each p is zero or 1;R^(h) is OH, NH₂ or --NHLysNH₂ ; and R^(i) is H or COCH₃.
 2. The complexof claim 1 wherein said first strand is a DNA strand.
 3. The complex ofclaim 1 wherein said first stand is a RNA strand.
 4. The complex ofclaim 1 wherein a majority of said ligands of at least one of saidsecond strand or third strand are pyrimidine bases.
 5. The complex ofclaim 4 wherein said pyrimidine bases are selected from thymine andcytosine.
 6. The complex of claim 1 wherein said nucleic acid strand isa purine rich strand.
 7. The complex of claim 1 wherein said interactionbetween said first and second strands occurs between heterocyclic basesof said first strand and ligands on said second strand.
 8. The complexof claim 1 wherein said interaction between said first and third strandsoccurs between heterocyclic bases of said first strand and ligands onsaid third strand.
 9. The complex of claim 1 wherein said interactionincludes formation of non-covalent bonds.
 10. The complex of claim 1wherein said interaction of ligands is via hydrogen bonding.
 11. Thecomplex of claim 1 wherein said interaction between said first andsecond strands occurs between heterocyclic bases of said first strandand ligands on said second strand;said interaction between said firstand third strands occurs between heterocyclic bases of said first strandand ligands on said third strand; and said interaction of ligands ofsaid second strand and heterocyclic bases of said first strand is viaWatson/Crick hydrogen bonding, and said interaction of ligands of saidsecond strand with heterocyclic bases of said first strand is via one ofWatson/Crick hydrogen bonding or Hoogsteen hydrogen bonding.